SUPPLEMENTAL MATERIALS

A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes:

A New Resource for Understanding Brain Development and Function

JD Cahoy, B Emery, A Kaushal, LC Foo, JL Zamanian, KS Christopherson, Y Xing, JL Lubischer, P Krieg, SA Krupenko, WJ Thompson, BA Barres

Supplemental Experimental Procedures The purification procedures are based on previously described dissociation (Huettner and Baughman, 1986; Segal et al., 1998) and immunopanning purification protocols for other cell types (Barres et al., 1988; Barres et al., 1992; Meyer-Franke et al., 1995). All aspects of the panning and FACS purification procedures are summarized below.

Preparation Of Mouse Forebrain Cell Suspensions

Six to eight mice from a wild-type litter (C57BL/6, Charles River, Wilmington, MA) or S100- EGFP transgenic litter (C57BL/6 x DBA (F1), Kosmos line, Zuo et al., 2004) were used. The forebrain was isolated by removal of the olfactory lobes, cerebellum, and midbrain/hindbrain structures by crude dissection, and the tissue was diced with a curved-blade surgical scalpel (Feather, Osaka, Japan, GRF-2976 #10). To isolate cerebral cortical gray matter astrocytes, the brain was sliced in 2-3

1 Page 1 of 58 mm coronal sections and the cerebral cortex was carefully dissected away from the ventral white matter tracks. This tissue was enzymatically dissociated to make a suspension of single cells, essentially as described by Huettner and Baughman (Huettner and Baughman, 1986; Segal et al., 1998). Briefly, the tissue was incubated at 33 °C for 80 minutes (90 minutes for animals P16 and older) in 20 ml of a papain solution (20 U/ml, Worthington, Lakewood, NJ, LS03126) prepared in dissociation buffer with EDTA (0.5 mM), and L-cysteine-HCl (necessary to activate the papain, 1 mM, Sigma, St. Louis, MO, C7880). The dissociation buffer contained Earle’s balanced salts (EBSS,

Sigma, E7510), D(+)-glucose (22.5 mM), NaHCO3 (26 mM), and DNase (125U/ml, Worthington,

LS002007) and requires careful equilibration with 5% CO2 and 95% O2 gas before use and during papain treatment. When dissociation buffer is exposed to room air during trituration, minimizing surface area and avoiding bubbles is essential to maintain the proper pH and cell health. After papain treatment to loosen contacts in the extracellular matrix, the tissue was washed with 3 x 4 ml dissociation buffer containing BSA (1.0 mg/ml, Sigma, A-8806) and ovomucoid (also known as Trypsin Inhibitor, 1.0 mg/ml, Roche Diagnostics Corporation, Indianapolis, IN, 109878) (inhibitor solution) and then mechanically dissociated by gentle sequential trituration using a 5 ml pipette with 5 x 4 ml fresh inhibitor solution to yield a suspension of single cells. Dissociated cells were layered on top of 12 ml of concentrated inhibitor solution (5 mg/ml BSA and 5 mg/ml ovomucoid) and harvested by centrifugation (140 x g for 5 minutes, 220 x g for 10 minutes when purifying OL lineage cells). This method routinely yielded ~15-20 million cells per mouse pup forebrain, with excellent cell health as determined by morphology and viability (>90% by trypan blue exclusion).

Preparation of Panning Plates Secondary antibodies (Jackson ImmunoResearch, West Grove, PA) included affinity-purified

goat anti-mouse IgG + IgM H+L chain (115-005-044), goat anti-mouse IgM μ-chain (115-005-02), and goat anti-rat IgG H+L chain (112-005-167). Primary antibodies included O4 and O1 hybridoma supernatant (mouse IgM, Bansal et al., 1989), anti-GalC hybridoma supernatant (mouse IgG, Ranscht et al., 1982), anti-MOG hybridoma supernatant (mouse IgG, from R. Reynolds, Imperial College,

2 Page 2 of 58 London, UK), A2B5 monoclonal antibody ascites (American Type Culture Collection, Rockville, MD)

and anti-PDGFR (rat IgG, monoclonal antibody CD140a clone APA5, BD Biosciences-Pharmingen, San Jose, CA, 558774). Petri dishes (150 x 15 mm, BD Biosciences-Falcon 351058) were incubated with 22 ml of tris

buffer solution (50 mM, pH 9.5) and 150 μg secondary antibody overnight at 4 oC. Each dish was then washed three times with 30 ml of Dulbecco's phosphate-buffered saline (DPBS, Invitrogen, Carlsbad, CA, 14287) and incubated with the corresponding primary antibodies (4 ml hybridoma supernatant or

10 μg purified antibody) diluted in a DPBS/0.2% BSA solution (12 ml total per dish) for at least 1 hour at room temperature. Lectin coated panning plates were prepared by adding 22 ml of DPBS and 50 μg of Bandeiraea Simplicifolia lectin I (BSL1, Vector Laboratories, Burlingame, CA, L-1100) and incubating at room temperature for 4 hours. All panning dishes were washed three times with 30 ml DPBS immediately before use.

Panning Purification Of Mouse Oligodendrocyte Lineage Cells Dissociated mouse forebrain was resuspended in 12 ml panning buffer (DBPS containing

0.02% BSA and 12.5 U/ml DNase) and filtered through a 20μm Nitex mesh (Sefar America Inc., Depew, NY, Lab Pak 03-20/14). In order to deplete microglia, the single-cell suspension was sequentially panned on four BSL1 panning plates, incubating each plate for 15 minutes at room temperature and then transferring nonadherent cells to the next panning plate. The cell suspension was

then sequentially incubated for 45 minutes on two PDGFR plates (to purify and deplete OPCs), one A2B5 plate (to deplete any remaining OPCs), two MOG plates (to purify and deplete myelinating

OLs), and one GalC plate (to purify the remaining PDGFR-, MOG-, OLs). The adherent cells on the

first PDGFR, MOG, and GalC plates were washed 8 times with 20 ml of DPBS to remove all antigen-negative nonadherent cells. For purification of RNA, the cells were lysed while still attached

to the panning plate with 2 ml Qiagen RLT lysis buffer with 1% -Mercaptoethanol, and total RNA was purified as described below. To confirm the purity of specific stages of OL development and myelination, purified cells were removed from the final panning plate by treating with trypsin (Sigma, 1,000 U/ml, T-4665) in 6

3 Page 3 of 58 ml Ca2+- and Mg2+-free EBSS (Irvine Scientific, Santa Ana, CA, 9208) for 10 minutes at 37 °C in a

10% CO2 incubator. The trypsin was then neutralized with 12 ml of Dulbecco's modified eagle medium (DMEM, Invitrogen, 11960-044) containing 20% fetal bovine serum (FBS, Invitrogen, 10437-028) and the cells were dislodged by gentle pipetting and harvested by centrifugation at 220 x g for 15 minutes. The cells were resuspended in culture media and transferred to poly-D-lysine (10

μg/ml in water, Sigma, P6407) coated 12 mm glass coverslips in 24-well tissue culture plates. Acutely purified OL lineage cells were cultured for 24 hours and then immunostained for stage specific-antigen expression. Purified OPCs were >95% NG2 positive and 0% MOG positive. Purified Myelin OLs were 100% MOG positive, >95% MBP positive, and 0% NG2 positive. Purified GalC OLs depleted of OPCs and Myelin OLs were <10% MOG positive and ~50% weakly NG2 positive, a reflection of their recent development as early OLs.

FACS Purification Of Mouse Astrocytes

Dissociated forebrain from S100-EGFP mice was resuspended in 12 ml panning buffer, filtered through Nitex mesh, and sequentially incubated on the following panning plates (to remove OLs because these cells also express EGFP): secondary antibody only plate to deplete microglia that stick nonspecifically via their Fc receptors (goat anti-mouse IgG + IgM, 15 minutes), O4 plate to

deplete OLs (20 minutes), PDGFR plate to deplete OPCs (30 minutes), and a second O4 plate to deplete any remaining OLs (30 minutes). This procedure was sufficient to deplete all OL lineage cells from animals P8 and younger, however, in older animals that had begun to myelinate, additional depletion of OLs and myelin debris was accomplished as follows. The nonadherent cells from the last O4 dish were harvested by centrifugation at 140 x g for 5 minutes, and then 20 million cells were resuspended in 5 ml of panning buffer containing GalC, MOG, and O1 supernatant (each diluted 1:30) and incubated for 15 minutes at room temperature. The cell suspension was washed by diluting with 40 ml panning buffer and centrifuging at 220 x g for 5 minutes, and then resuspended in 4ml of

panning buffer containing 20 μg donkey anti-mouse APC (eBioscience, San Diego, CA, 17-4012-82) for 15 minutes. The cells were washed and resuspended in 3 ml of panning buffer containing 1 μg/ml propidium iodide (PI, Sigma-Aldrich, P4864) for FACS purification.

4 Page 4 of 58 EGFP positive astrocytes were purified by fluorescence activated cell sorting (FACS) using a FACS Vantage SE sorter (Becton Dickinson) with CellQuest software, and resulting data were analyzed with Flowjo (Treestar, Ashland, OR). Dead cells were gated out using high PI staining and forward light scatter. Astrocytes were identified based on high EGFP fluorescence and negative APC fluorescence from indirect immunostaining for OL markers GalC, MOG, and O1. Cells were sorted twice and routinely yielded >99.5% purity based on reanalysis of double sorted cells.

FACS Purification Of Neurons EGFP negative cells were the remaining forebrain cells after microglia, OLs, and astrocytes had been removed, and were primarily composed of neurons, and to a lesser extent, endothelial cells (we estimate < 4% endothelial cells at P7 and < 20% endothelial cells at P17). Other extremely minor potential constituents include meningeal cells, ependymal cells, and pericytes. EGFP negative cells

from S100-EGFP dissociated forebrain were FACS purified in parallel with astrocyte purification, and were sorted based on their negative EGFP fluorescence. Cells were sorted twice and routinely yielded >99.9% purity. FACS purification of EGFP negative cells yielded an enriched neuron sample free of microglia, OLs, and astrocytes. In independent preparations, the EGFP negative cell population was additionally depleted of

endothelial cells and pericytes by sequentially labeling with biotin-BSL1 lectin (12.5μg, Vector Laboratories, B-1105) and Streptavidin-APC (10 μg, eBioscience, 17-4317-82) while also labeling for OL markers as described above. Cells were sorted twice and routinely yielded >99.9% purity. FACS purification of EGFP negative and APC negative cells yielded a highly enriched neuron sample free of endothelial cells, pericytes, microglia, OLs, and astrocytes.

Preparation Of Mouse Astroglia Preparation of astroglia cultures was as follows. Cortices were isolated by crude dissection of six P1 mice from a S100-EGFP transgenic litter, meninges were removed, and the tissue was chopped using a curved-blade surgical scalpel. This tissue was enzymatically dissociated to make a suspension of single cells. Briefly, the tissue was incubated at 33 °C for 75 minutes in 20 ml of a papain solution prepared in dissociation buffer containing EDTA and L-cysteine.

5 Page 5 of 58 After papain treatment the tissue was washed with 3 x 4 ml of DPBS containing BSA (1.5 mg/ml), ovomucoid (1.5 mg/ml), and DNase (125U/ml) (ovomucoid inhibitor). The tissue was then mechanically dissociated by gentle sequential trituration using a 5 ml pipette with 5 x 4 ml fresh ovomucoid inhibitor to yield a suspension of single cells. Dissociated cells were harvested by centrifugation at 220 x g for 10 minutes. Cells were resuspended in 12 ml of concentrated ovomucoid inhibitor (5 mg/ml BSA and 5 mg/ml ovomucoid) and harvested by centrifugation. Dissociated cells were resuspended in 15 ml of astroglia media and plated in 75 cm2 tissue culture flasks (BD Falcon,

353136) that were pre-coated with PDL (10 μg/ml in water, Sigma, P6407) at a density of 25-30 million cells per flask. Astroglia media contained Dulbecco's modified eagle medium (DMEM, Invitrogen, 11960-044), 10% fetal bovine serum (FBS, Invitrogen, 10437-028), 2 mM glutamine (Invitrogen, 25030-081), 1 mM Na pyruvate (Invitrogen, 11360-070), 5 μg/ml N-Acetyl-L-cysteine (NAC, Sigma, A8199), 5 μg/ml insulin (Sigma, I6634), 100 U/ml penicillin-streptomycin (Invitrogen, 15140-122), and 10 μM hydrocortisone (Sigma, H0888). When plated under these conditions most cells including neurons and mature astrocytes die, but a small percentage of immature cells are able to survive and proliferate. After 3 days the astroglia layer was confluent and the flask was rinsed once with DPBS and then shaken 3 times with DPBS containing phenol red to remove contaminating OL progenitor cells (OPCs), and fresh astroglia media was added. OPCs grow on top of the single cell monolayer of astroglia, so they can be removed by vigorous shaking of the sealed tissue culture flask. After 2 days,

10 μM cytosine arabinoside (AraC, Sigma, C1768) was added to kill dividing cells such as fibroblasts and microglia; the underlying monolayer of astrocytes is not affected by the antimitotic because astroglia divide slowly in dense cultures due to contact inhibition. After 48 hours the astroglia were trypsinized off and plated onto PDL-coated 15 cm tissue culture dishes at 2 million cells per dish in astroglia media. For preparation of astroglia grown in serum free media, confluent cultures of astroglia prepared as described above were washed three times with DPBS and 20 ml serum-free media was added. Serum-free medium, modified from Bottenstein and Sato (1979), contained neurobasal (Invitrogen,

21103-049), bovine serum albumin (100 μg/ml, BSA, Sigma, A4161), selenium (40 ng/ml, Sigma,

6 Page 6 of 58 S5261), putrescine (16 μg/ml, Sigma, P5780), transferrin (100 μg/ml, Sigma, T1147), progesterone (60 ng/ml, Sigma, P8783), triiodo-thyronine (40 ng/ml, Sigma, T6397), penicillin-streptomycin (100U/ml),

insulin (5 μg/ml) NAC (5 μg/ml), pyruvate (1 mM), and glutamine (2 mM).

RNA Purification, Labeling, And Hybridization Total RNA was isolated from acutely purified cells with the RNeasy micro kit (Qiagen, Valencia, CA) using Qiashredder columns for cell lysis and Qiagen on-column DNase treatment to remove any contaminating genomic DNA. The integrity of RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara , CA), and RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Rockland, DE). Biotinylated cRNAs for hybridization to Affymetrix 3'-arrays were prepared from 30-50 ng total RNA using the Affymetrix two-cycle target labeling assay with spike in controls (Affymetrix Inc., Santa Clara, CA, 900494). Labeled-cRNA was fragmented and hybridized to Mouse Genome 430 2.0 Arrays (3'-arrays, Affymetrix, 900495) following the manufacturer’s protocols. Biotinylated sense-strand DNA for hybridization to Affymetrix exon-arrays was prepared with

the Affymetrix GeneChip whole transcript sense target labeling assay, using either the standard 1 μg total RNA method or the reduced starting material 100 ng method according to the manufacturer’s

protocols (Affymetrix, 900854). Briefly, for samples with 1 μg of total RNA, ribosomal RNA reduction was performed using the RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen, K1550-01); ribosomal RNA reduction was not performed for the 100 ng reduced starting material samples. The labeling assay yielded labeled single-stranded DNA that was fragmented and hybridized to Affymetrix Mouse Exon 1.0 ST Arrays (exon-arrays) according to manufacturer’s protocols.

Data Normalization and Analysis Raw image files were processed using Affymetrix GCOS 1.3 software to calculate individual probe cell intensity data and generate CEL data files. Using GCOS and the MAS 5.0 algorithm, intensity data was normalized per chip to a target intensity TGT value of 500, and expression data and present/absent calls for individual probe sets were calculated. symbols and names for data analyzed with the MAS 5.0 algorithm were from the Affymetrix NetAffx Mouse430_2 annotations file

7 Page 7 of 58 (http://www.affymetrix.com/support/technical/byproduct.affx?product=moe430-20). Quality control was performed by examining raw DAT image files for anomalies, confirming each GeneChip array had a background value less than 100, monitoring that the percentage present calls was appropriate for the cell type, and inspecting the poly(A) spike in controls, housekeeping , and hybridization controls to confirm labeling and hybridization consistency. In addition, the quantity of RNA purified per cell, the labeling efficiency for each cycle of amplification, and the fragmentation efficiency were carefully monitored. CEL data files were also independently analyzed using the dChip algorithm. values were normalized and modeled across arrays using the dChip software package with invariant- set normalization and a PM model (www.dchip.org, Li and Wong, 2001). Gene names were assigned to probe sets using the annotate (Gentleman) package and the mouse4302 library in Bioconductor. Global hierarchical clustering (Supplemental Figure S3 and S10) was also performed using the dChip package. Gene filtering was performed to select probe sets that were consistently expressed in at least one cell type. The 29 samples were grouped into 9 sample types: Astros P7-P8, Astros P17, Astros P17g, Neurons P7, Neurons P17, Neurons P7n/P17n, OPCs, GalC-OLs, and MOG-OLs. We identified 20,932 of the 45,037 probe sets that were consistently expressed in at least one of the nine cell types, where consistently expressed was defined as being called present and having a MAS 5.0 intensity level greater than 200 in at least two-thirds of the samples in the cell type. This cell-type specific filtering was performed to ensure we did not eliminate probe sets that were expressed in only one cell type. When applying our filter, we considered all probe sets except the AFFX control probe sets. After filtering, we got 20,932 probe sets that met our filter. We then mapped these probe sets to gene identifiers using the mouse4302 library in Bioconductor; probe sets that could not be mapped were not included. This analysis led to identification of 12,416 unique genes. In the case that multiple probe sets mapped to one gene, we selected the probe set with the greatest standard deviation of expression values. The Significance Analysis of Microarrays (SAM) method (Tusher et al., 2001) was used to determine genes that were significantly differentially expressed between different cell types.

8 Page 8 of 58 Significance thresholds were set to find genes with fold-change greater than 1.5 and false discovery rate (FDR) less than 1%. The analysis was performed using the samr package in R. When using SAM analysis to construct our enriched genes lists, for genes with multiple probe sets, we listed the probe set with greatest fold change. Ingenuity Pathways Analysis was used for functional analyses of genes enriched in astrocytes, neurons, and OLs (Redwood City, CA). Complete lists of all cell type enriched genes appear in the Supplemental Tables. Genes names are listed with the Affymetrix probe set ID that can be used to identified the original mRNA region targeted, individual probes used, and the current gene assignment by query at NetAffx (http://www.affymetrix.com/analysis/index.affx). Clustering was performed using the hclust method with complete linkage in R. Expression values were transformed for clustering by computing a mean expression value for the gene using those samples in the corresponding SAM statistical analysis, and then subtracting the mean from expression

intensities. In order to preserve the log2 scale of the data, unless otherwise indicated, no normalization by variance was performed. Plots were created using the gplots package in R (http://cran.r- project.org/src/contrib/Descriptions/gplots.html). The Bioconductor software package (Gentleman et al., 2004) was used throughout the expression analysis, with specific use of the affy (Irizarry et al., 2006), genefilter (Gentleman et al.), annotate (Gentleman), and mouse4302 (Liu et al.) libraries. Exon-array expression indexes were calculated using the GeneBASE program (Kapur et al., 2007). Briefly, the program employs a sequence-specific background model to correct background

intensities of exon-array probes (Kapur et al., 2007), followed by an iterative probe selection procedure (Xing et al., 2006) that selects a subset of highly correlated probes of a gene for expression index computation. To compare 3’-array data to exon-array data, we mapped the 3’-array probe sets to mouse exon-arrays using a mapping file provided by Affymetrix (http://www.affymetrix.com/ Auth/analysis/downloads/na20/exon/MOE430-MoEx1_0st-transcript-cluster-mapping.zip) and our manual query of NetAffx (http://www.affymetrix.com/analysis/ index.affx).

Immunohistochemistry

9 Page 9 of 58 Mice or rats were perfused with DPBS followed by 4% paraformaldehyde and then post-fixed in 4% paraformaldehyde overnight at 4°C. Brains were cryoprotected overnight at 4°C in 15% sucrose, frozen in 1 part Tissue Tek O.C.T. and 2 parts 30% sucrose, and 10μm sections were cut. Sections were blocked in 50% goat serum with 0.4% Triton-X100 in PBS for 45 minutes at room temperature, and primary antibody diluted in antibody buffer (1% BSA, 150mM NaCl, 50mM Tris, 100mM L-Lysine, 0.04% Azide, pH 6.4) was added over two nights at 4°C. Sections were incubated with Molecular Probes Alexa Fluor 594 and 488 goat anti-rabbit and anti-mouse 594 IgG highly- crossed adsorbed secondary antibodies at 1:500 (Molecular Probes-Invitrogen, A11029, A11037, A11032) for two hours at room temperature. Rabbit serum against rat Aldh1L1 (Krupenko and Oleinik, 2002) was used at 1:1000. Mouse monoclonal anti-GFAP (used for rat IHC, 1:1000, Sigma G-3893), rabbit polyclonal anti-GFAP (used for mouse IHC, 1:5000, Dako, Carpinteria, CA Z0334), mouse monoclonal anti- isotype III (1:1000, Sigma T8660), mouse monoclonal anti-NG2 chondroitin sulfate proteoglycan (1:400, Chemicon-Millipore, Billerica, MA, MAB5384), mouse monoclonal anti-MBP (1:100, Chemicon MAB382), and mouse monoclonal anti-APC (CC-1, 1:100, Calbiochem-EMD Chemicals, San Diego, CA, OP80) were used.

In situ hybridization PCR fragments of approximately 1 kb were generated from mouse brain cDNA using the

following primers: Nov: ggaagccaccgtaggagttgaa and gcattgggagtttttggttcag, Actl6b: gaactcccatcccatacctctc and catcgtctgcctcctgcttttt, Tmem130: tgtcgcctccccactgtcatct and catcgtctgcctcctgcttttt, Brunol4: ggaggctacctgaatcccatgg and ggcagcagcatctcctcctagt, Tmem125: gggtaaccatagcctggactaa and cccgagagtcactctctgaaga, Gpr62: cgcggctcacccctttctctat and ccccaaagaaccccagtctgaa. PCR fragments were subcloned into the pCR-II-TOPO vector and digoxigenin-labeled single-stranded antisense riboprobes were prepared by transcription of the linearized plasmid using either T7 or Sp6 RNA polymerases and DIG RNA labeling kit (Roche) as per manufacturer’s instructions. In situ hybridizations were performed essentially as described (Schaeren-

Wiemers and Gerfin-Moser, 1993), with some modifications for postnatal tissue. Briefly, 10 μm fresh- frozen sections of P16 mouse brain were air-dried and fixed in 4% paraformaldehyde for 10 minutes.

10 Page 10 of 58 After washing with PBS for 3 x 10 minutes, sections were acetylated by incubation in 0.1 M triethanolamine-HCl, 0.25% acetic anhydride for 10 min. After washing with PBS for 3 x 5 minutes, hybridization was performed with probes at concentrations of 200–500 ng/ml in a hybridization

solution (50% formamide, 5x SSC, 5x Denhardt’s, 250 μg/ml bakers yeast RNA, and 100 μg /ml salmon sperm DNA) at 72 °C for 16 h. After hybridization, slides were washed once in 0.2x SSC at 72 °C for 30 minutes, once in 0.2x SSC at room temperature for 5 min, once in buffer B1 (0.1M Tris-HCl pH7.5, 150mM NaCl) for 5 minutes, incubated in B1 with 10% normal goat serum for 1 hour, and then incubated in B1 with 1% normal goat serum and 1:5000 anti-digoxygenin alkaline-phosphatase conjugated antibody (Roche) overnight at 4 °C. Slides were then washed 3 x 5 minutes in B1,

equilibrated in buffer B3 (0.1M Tris-HCl pH 9.5, 100mM NaCl, 50mM MgCl2) for 15 minutes and then developed in B3 with 0.24 mg/ml levamisole 0.375 μl/ml NBT and 3.5 μl BCIP/ml (Roche) until a color reaction was visible. The reaction was stopped by washing once in TBST and four times in

water, and slides were coverslipped with glycergel mounting medium (Dako).

Supplemental Figure and Table Legends

Supplemental Figure S1. Purity of FACS purified EGFP positive astrocytes after single and double sorting. (a) Cells from wild-type control mice show no fluorescence. (b) After depleting OLs and microglia by immunopanning, astrocytes from postnatal day 7 S100-EGFP transgenic mice show

strong EGFP fluorescence and as expected represent ~35% of the cells at this age. (c) The purity of EGFP positive astrocytes after the first sort is 97.7% and (d) double sorting increases the purity to 99.8%. The x-axis is a log scale of the level of EGFP fluorescence, the y-axis is the number of cells at each level of fluorescence; only living (propidium iodide negative) cells are shown.

Supplemental Figure S2: Unsupervised hierarchical clustering of expressed genes. Unsupervised hierarchical gene clustering and heat map of 12,416 significantly expressed CNS genes. Each row represents a gene whose expression level in each sample (column) is compared to the average

11 Page 11 of 58 expression level in all samples. This ratio is displayed using a colored scale based on the standard deviation of aggregate samples: greater than average gene expression, red; less than average gene expression, blue; or the same level of expression, white.

Supplemental Figure S3: Astrocyte, oligodendrocyte, and neuron-specific genes. The top 250 cell type enriched genes are plotted on a heat map to show genes enriched in astrocytes (green bar), neurons (yellow bar), and OLs (red bar). This figure demonstrates that the number of highly enriched genes is much greater than the 40 genes shown in Figure 3. Genes names and fold enrichment are listed in Supplemental Tables S4-S6.

Supplemental Figure S4: Comparison of the Allen Brain Atlas astrocyte-enriched gene list with 3’-array expression levels in highly purified CNS cells. Genes identified as putative astrocyte- enriched genes using in situ hybridization in Lein et al., 2007 are plotted on a heat map showing the expression levels in highly purified CNS cells as determined by 3’-arrays. Each individual gene

expression level was normalized and plotted on a log2 color scale with blue representing low

expression and red representing high expression. The fold enrichment can be estimated from the log2 color bar scale, for example, the change from medium blue (-2) to red (2) represents a 16-fold difference in expression level. 71% of the genes identified in the Allen Brain Atlas as being astrocyte- enriched are greater than 1.5-fold astrocyte-enriched by 3’-arrays, although only 21% display an astrocyte-specific pattern (greater than 20-fold enriched). Some genes show expression in other CNS cell types: Sox8, Dip2a, Gsn, Matn4, Sept4 are OL-enriched, Atp13a5 and Cldn5 are enriched in

endothelial cells, and C1qa and Selpl are microglia-enriched (C1qa and Selpl expression in microglia is ~200-fold higher than in OLs; the expression in OLs is near background levels). 6 genes identified in Lein et al., 2007, not present or without detectable expression in 3’-arrays, have been omitted.

Supplemental Figure S5: Comparison of the Allen Brain Atlas oligodendrocyte-enriched gene list with 3’-array expression levels in highly purified CNS cells. Genes identified as putative OL- enriched genes using in situ hybridization in Lein et al., 2007 are plotted on a heat map showing the expression levels in highly purified CNS cells as determined by 3’-arrays. Each individual gene

12 Page 12 of 58 expression level was normalized and plotted on a log2 color scale with blue representing low

expression and red representing high expression. The fold enrichment can be estimated from the log2 color bar scale, for example, the change from medium blue (-2) to red (2) represents a 16-fold difference in expression level. 81% of the genes identified in the Allen Brain Atlas as being OL- enriched are greater than 1.5-fold OL-enriched by 3’-arrays, although only 20% display an OL-specific pattern (greater than 20-fold enriched). Some genes show expression in other CNS cell types: S100a16, Qk, Npc1, Sepp1, Csrp1, Daam2, Serpinb1a, Map2k6, Fapb5, Car2, and Tegt are enriched in astrocytes. 3 genes identified in Lein et al., 2007, not present or without detectable expression in 3’- arrays, have been omitted.

Supplemental Figure S6: Comparison of the Allen Brain Atlas neuron-enriched gene list with 3’- array expression levels in highly purified CNS cells. Genes identified as putative neuron-enriched genes using in situ hybridization in Lein et al., 2007 are plotted on a heat map showing the expression levels in highly purified CNS cells as determined by 3’-arrays. Each individual gene expression level

was normalized and plotted on a log2 color scale with blue representing low expression and red

representing high expression. The fold enrichment can be estimated from the log2 color bar scale, for example, the change from medium blue (-2) to red (2) represents a 16-fold difference in expression level. 82% of the genes identified in the Allen Brain Atlas as being neuron-enriched are greater than 1.5-fold neuron-enriched by 3’-arrays, although only 11% display a neuron-specific pattern (greater than 20-fold enriched). Some genes show expression in other CNS cell types: Chst1, Egr1, and Alcam

are enriched in astrocytes and Gria4, Myo5a, and Cyfip2 are enriched in OLs. 4 genes identified in Lein et al., 2007, not present or without detectable expression in 3’-arrays, have been omitted.

Supplemental Figure S7: GENSAT expression patterns for pan-astrocyte marker Aldh1L1 and white-matter astrocytes marker GFAP. Expression of EGFP in GENSAT Aldh1L1-EGFP BAC mouse brain and GENSAT GFAP-EGFP BAC mouse brain, showing predominant white matter expression in the case of the GFAP-EGFP BAC, but pan-astrocyte expression of EGFF in the case of the Aldh1L1-EGFP BAC. Images courtesy of GENSAT (Heintz, 2004).

13 Page 13 of 58 Supplemental Figure S8: FACS characterization of the BAC Aldh1L1-EGFP mice. Acutely dissociated forebrain of BAC Aldh1L1-EGFP mice was immunostained anti-O4 monoclonal antibody labeling OLs. The EGFP positive astrocytes are well separated from the EGFP negative cells, which will allow for good FACS purification of EGFP positive astrocytes. The BAC Aldh1L1-EGFP does not label OLs, which makes this a improved mouse for FACS purifying astroctes compared to the

S100 mice.

Supplemental Figure S9: Genes upregulated and downregulated during astrocyte and oligodendrocyte development. The top 250 genes most downregulated during astrocyte development (a, light green bar) and OL development (b, orange bar) and the top 250 genes most upregulated during astrocyte development (c, dark green bar) and OL development (d, red bar). The upregulated and downregulated genes are plotted on a heat map to show the cell type specific gene expression patterns in all CNS cell types at different developmental stages. This figure shows that the pattern of cell type expression for the top 250 is very similar to the patter for the 40 genes shown in Figure 6, such that few genes most downregulated during development (a, b) are expressed in a cell type specific pattern, while many genes most upregulated during development (c, d) are expressed in a cell type specific pattern. A complete list of genes appears in Supplemental Tables S15-S18.

Supplemental Figure S10: Cultured astroglia dendrogram and sample clustering. Hierarchical clustering of cultured astroglia samples along with highly purified in vivo CNS cell types from different developmental stages shows samples cluster into three main branches representing astrocytes, neurons, and OLs, with cultured astroglia branching off of the in vivo astrocyte branch. The similarity of gene expression between different samples is represented by the vertical distances on each branch of the dendrogram. Although the cultured astroglia cluster with the in vivo astrocytes branch, cultured astroglia branch off at a similar level at which OPC branch away from OLs, and P1 astrocytes branch away from P7 and P17 astrocytes. This suggests that although cultured astroglia are more similar to in vivo astrocytes than to in vivo neurons or OLs, large differences between cultured astroglia and in vivo astrocytes remain.

14 Page 14 of 58 Supplemental Figure S11: Cultured astroglia unsupervised hierarchical clustering of the 12,416 CNS expressed genes. Cultured astroglia samples and in vivo purified CNS samples were analyzed by unsupervised hierarchical gene clustering of 12,416 significantly expressed CNS genes. It can be seen that the gene expression patterns of cultured astroglia most resemble that of in vivo astrocytes. Important differences remain, such as those genes expressed higher in cultured astroglia compared to in vivo astrocytes (teal bar), genes expressed at higher levels in cultured astroglia, young astrocytes, and OPCs, but not in vivo mature astrocytes, OLs, or neurons (light green bar), and those genes expressed at lower and less consistent levels in cultured astroglia than in mature astrocytes in vivo (green bar). Each row represents a gene whose expression level in each sample (column) is compared to the average expression level in all samples. This ratio is displayed using a colored scale based on the standard deviation of aggregate samples: greater than average gene expression, red; less than average gene expression, blue; or the same level of expression, white.

Supplemental Table S1: Gene expression levels for well-described markers of astrocytes, oligodendrocytes, neurons, microglia, and endothelial cells. Representative genes specifically expressed by (a) astrocytes, (b) OLs, (c) neurons, (d) microglia, and (e) endothelial cells demonstrates

high purity of each cell type. (f) Endogenous S100 is expressed by astrocytes and OLs, but not in neurons. Gene expression levels are calculated by the MAS 5.0 algorithm. Absent/present detection calls are calculated using probe-match probe-mismatch pairs, and expression values in black have been called present in at least 2/3 of samples, while those that appear in gray have been called absent or marginal. Genes with expression below 100 are generally called absent, although the absent/present calculation is independent of the gene expression value calculation. When a gene is represented by more than one probe set, the probe set with the best sensitivity and range is listed. Expression levels for microglia are from mixed microglia-OPC PDGFR panning that contain an estimated 1/3 microglia (BE and BAB unpublished data), and expression levels for brain endothelial cells are from FACS purified endothelial cells (R. Daneman and B. A. Barres, unpublished data).

Supplemental Table S2: GeneChip file name, age, sample type, and sample name and grouping for statistical analysis by SAM. (a) Each GeneChip Array experiment is listed by the file name that

15 Page 15 of 58 can be used to reference the original raw data file. (b) The age is the number of days after birth (postnatal day) for acutely isolated in vivo samples and is the number of days in culture for in vitro astroglia samples (all cultured astroglia were prepared from a litter of pups 1 day after birth). All

samples were prepared from S100-EGFP transgenic mouse litters unless noted as prepared from wild- type C57BL6 mouse litters. (c) Sample type describes the final step of the purification: FACS purified EGFP positive astrocytes (positive) or EGFP negative neurons (negative), the Neg(Bsl1) sample is FACS purified EGFP negative cells that have been further depleted of endothelial cells with BSL1; panning with monoclonal antibodies against PDGFR for OPCs, GalC for OLs, or MOG for myelinating OLs; harvesting cultured astroglia RNA by lysing the cells directly on the culture dish (culture), preparing a single cell suspension of cultured astroglia and FACS purifying all live cells (FACS all), or FACS purifying cultured astroglia with high levels EGFP fluorescence (FACS pos); the forebrain sample was prepared by purifying total RNA from freshly dissected forebrain and represents the brain before cell type purification. (d) Sample names used in the figures, tables, and text. (e) Samples are grouped as described in the columns and genes statistically enriched in each sample type are identified by performing SAM statistical analysis using a false discovery rate (FDR) threshold of 1% and a 1.5-fold expression difference threshold: astrocytes (A), OLs (O), and neurons (N) are compared to the other purified cell types (X); young astrocytes (AY) are compared to old astrocytes (AO); OPCs are compared to myelinating OLs (MOG-O); OLs remaining after OPC and MOG-O depletion (GalC-O) are compared to MOG-O; in vivo mature P17 astrocytes (AO) are compared to in vitro cultured astroglia (Cult). John_14C, John_14D, and John_14F, and John_19C, John_19D, and John_19E each represent identical biological samples and therefore only one sample from each is included in the SAM analysis. Samples 39-48 were assayed using exon-arrays, using either 100 ng

labeling or 1 μg labeling. Exon-array samples that used the same pool of starting total RNA as was used for 3’-array labeling are noted.

Supplemental Table S3: 3’-Array master data table of 20,932 probe sets with significant expression in the CNS. The expression levels of 20,932 probe sets that show significant expression in at least one purified CNS cell type on Affymetrix 3’-arrays were calculated using the MAS 5.0

16 Page 16 of 58 algorithm (a) and the dChip algorithm (b). (a) Affymetrix gene expression levels calculated by the MAS 5.0 algorithm with normalization to 500 for the level of an average gene. Replicate samples are grouped and the geometric mean was calculated and appears in the table. Absent/present detection calls are calculated using probe-match probe-mismatch pairs and expression values in black have been called present in at least 2/3 of samples while those that appear in gray have been called absent or marginal. Genes with expression below 100 are generally called absent, although the absent/present calculation is independent of the gene expression value calculation. Gene symbols and names are from

the Affymetrix NetAffx Mouse430_2 annotations file. (b) dChip gene expression levels (log2) were calculated from CEL files that were normalized and modeled across arrays using the dChip software package with invariant-set normalization and a PM model. Replicate samples are grouped and the

average log2 expression value was calculated and appears in the table. Gene names were assigned to probe sets using the annotate (Gentleman) package and the mouse4302 library in Bioconductor. Gene expression values from dChip were used for statistical analysis and fold change enrichment calculations. The Affymetrix probe set ID can be used to identified the original mRNA region targeted, individual probes used, and the current gene assignment by query at NetAffx (http://www.affymetrix.com/analysis/index.affx).

Supplemental Table S4: Genes statistically enriched in astrocytes. Genes enriched greater than 1.5-fold in astrocytes are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 2,618

astrocyte-enriched genes that are represented by 3,898 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set; other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S5: Genes statistically enriched in oligodendrocytes. Genes enriched greater than 1.5-fold in OLs are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 2,228 OL-enriched genes that are represented by 3,306 probe sets. Genes represented by multiple probe sets

17 Page 17 of 58 are listed with the largest difference probe set; other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S6: Genes statistically enriched in neurons. Genes enriched greater than 1.5- fold in neurons are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 2,036 neuron- enriched genes that are represented by 2,820 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set; other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S7: Comparison of cell type enriched genes from 3’-arrays to exon-arrays validates cell type specific gene expression. We compared our lists of the top 40 cell type specific genes based on 3’-array data with exon-array data to confirm these genes are cell type specific. We first mapped the top 40 astrocyte, OL, and neuron-enriched genes from the 3’-array probe sets (a total of 120 probe sets) to mouse exon-arrays using a mapping file provided by Affymetrix (http://www.affymetrix.com/Auth/analysis/downloads/na20/exon/MOE430-MoEx1_0st-transcript- cluster-mapping.zip) and our manual query of NetAffx (http://www.affymetrix.com/analysis/ index.affx). Of the 120 3’-array probe sets, 101 were mapped unambiguously to unique exon-array gene identifiers that were represented by core probe sets with defined expression index computations. Of these 101 genes, 92 (91%) were at least 15-fold enriched in their respective cell type according to the exon-array expression indexes, 99 (99%) were at least 5-fold enriched, and the minimal exon-array fold change was 3.9-fold. These exon-array results which use different labeling methods and probe selected regions provide strong independent evidence for CNS cell type specific genes discovered from the 3’-arrays.

Supplemental Table S8: Exon-array master data table of genes expression in the CNS. Table of gene expression levels from Affymetrix exon-arrays. Included are the 17,213 genes that have defined transcript level probe selection and expression indexes (Xing et al., 2006).

18 Page 18 of 58 Supplemental Table S9: Selected astrocyte metabolic pathway enzymes. Selected metabolic enzymes that are enriched in astrocytes. See Table 1d-f for those metabolic pathways that are significantly enriched in astrocytes.

Supplemental Table S10: DNA binding enriched in or expressed at high levels in astrocytes. List of DNA binding proteins that may have important roles in controlling astrocyte gene expression. Expression levels from exon-arrays (probesetID_exon) are listed when the 3’-array (probesetID_at) expression level was too low to be informative.

Supplemental Table S11: Selected astrocyte expressed receptors. Astrocyte expressed receptors that may be important mediators of cell-cell interaction in the developing and functioning CNS, and

also in CNS disease. PDGFR is enriched in astrocytes, and also is important in Schwann cell development and function (Oya et al., 2002). Ryk is a coreceptor with Frizzled for Wnt ligands (Lu et al., 2004).

Supplemental Table S12: Selected astrocyte-enriched genes. Gene expression levels of selected astrocyte-enriched genes.

Supplemental Table S13: Astrocyte expressed signals that might control endothelial development and motility. Selected factors expressed by astrocytes that might mediate cell-cell signaling between astrocytes and endothelial cells. Angiomotin (Amot) is believed to play a role in endothelial cell motility (Bratt et al., 2005). Mfge8, also known as lactadherin, is believed to promote VEGF- dependent neovascularization (Silvestre et al., 2005).

Supplemental Table S14: Creatine and Carnitine synthetic enzymes statistically enriched in oligodendrocytes. List of synthetic enzymes for creatine and carnitine expressed by OLs. Gatm and Aldh9a1 display preferential expression in mature myelinating OLs.

Supplemental Table S15: Genes statistically enriched in developing astrocytes. Genes statistically enriched greater than 1.5-fold in immature astrocytes (P7-P8) compared to mature astrocytes (P17) are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are

19 Page 19 of 58 statistically different by SAM analysis with a FDR threshold <1%. There are 1,135 immature astrocyte-enriched genes represented by 1,460 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set, other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S16: Genes statistically enriched in mature astrocytes. Genes enriched greater than 1.5-fold in mature astrocytes (P17) compared to immature astrocytes (P7-P8) are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 1,133 mature astrocyte-enriched genes that are represented by 1,508 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set; other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S17: Genes statistically enriched in OPCs. Genes enriched greater than 1.5-

fold in OPCs compared to myelinating OLs (MOG+ OLs) are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 4,583 OL-enriched genes that are represented by 6,514 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set, other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S18: Genes statistically enriched in myelinating OLs compared to OPCs.

Genes enriched greater than 1.5-fold in myelinating OLs (MOG+ OLs) compared to OPCs are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 2,069 Myelin OL-enriched genes that are represented by 3,059 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set; other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S19: Genes statistically enriched in MOG positive myelinating OLs compared to MOG negative OLs. Genes enriched greater than 1.5-fold in myelinating OLs (MOG+

20 Page 20 of 58 OLs) compared to newly formed OLs (GalC+ MOG- OLs) are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 1,209 myelinating OLs-enriched genes that are represented by 1,724 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set; other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S20: Genes statistically enriched in astrocytes in vivo compared to cultured astroglia. Genes enriched greater than 1.5-fold in astrocytes in vivo compared to cultured astroglia are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 2,819 in vivo astrocyte- enriched genes that are represented by 4,192 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set; other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S21: Genes statistically enriched in cultured astroglia compared to in vivo astrocytes. Genes enriched greater than 1.5-fold in cultured astroglia compared to in vivo astrocytes are listed along with their fold change enrichment and the Affymetrix probe set ID. All genes are statistically different by SAM analysis with a FDR threshold <1%. There are 2,103 cultured astroglia- enriched genes that are represented by 2,769 probe sets. Genes represented by multiple probe sets are listed with the largest difference probe set; other probe sets are found on the master data table, Supplemental Table S3.

Supplemental Table S22: Genes in the Notch Signaling pathway. List of Notch signaling genes expressed in different CNS cell types. The downstream Notch effector genes Hes1, Hes5, Hey1, and Hey2 are enriched in both developing and mature astrocytes, suggesting selective activation of Notch signaling in astrocytes in vivo. Dll4, Jag1, and Jag2 are expressed by endothelial cells (R. Daneman and B. A. Barres, unpublished data).

REFERENCES

21 Page 21 of 58 Bansal R, Warrington AE, Gard AL, Ranscht B, Pfeiffer SE (1989) Multiple and novel specificities of monoclonal antibodies O1, O4, and R-mAb used in the analysis of oligodendrocyte development. J Neurosci Res 24:548-557.

Barres BA, Silverstein BE, Corey DP, Chun LL (1988) Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron 1:791- 803.

Barres BA, Hart IK, Coles HS, Burne JF, Voyvodic JT, Richardson WD, Raff MC (1992) Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70:31-46.

Bottenstein JE, Sato GH (1979) Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A 76:514-517.

Bratt A, Birot O, Sinha I, Veitonmaki N, Aase K, Ernkvist M, Holmgren L (2005) Angiomotin regulates endothelial cell-cell junctions and cell motility. J Biol Chem 280:34859-34869.

Gentleman R annotate: Annotation for microarrays. In, R package version 1.12.1. Edition.

Gentleman R, Carey V, Huber W genefilter: genefilter: methods for filtering genes from microarray experiments. In, R package version 1.12.0. Edition.

Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80.

Heintz N (2004) Gene expression nervous system atlas (GENSAT). Nat Neurosci 7:483.

Huettner JE, Baughman RW (1986) Primary culture of identified neurons from the visual cortex of postnatal rats. J Neurosci 6:3044-3060.

Irizarry RA, Gautier L, Bolstad BM, Miller C, Astrand wcfM, Cope LM, Gentleman R, Gentry J, Halling C, Huber W, MacDonald J, Rubinstein BIP, Workman C, Zhang J (2006) affy: Methods for Affymetrix Oligonucleotide Arrays. In, R package version 1.12.2. Edition.

Kapur K, Xing Y, Ouyang Z, Wong WH (2007) Exon array assessment of gene expression. Genome Biol 8:R82.

Krupenko SA, Oleinik NV (2002) 10-formyltetrahydrofolate dehydrogenase, one of the major folate enzymes, is down-regulated in tumor tissues and possesses suppressor effects on cancer cells. Cell Growth Differ 13:227-236.

Lein ES, Hawrylycz MJ, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445:168-176.

Li C, Wong WH (2001) Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application. Genome Biol 2:RESEARCH0032.

Liu TY, Lin CW, Falcon S, Zhang J, MacDonald JW mouse4302: Affymetrix Mouse Genome 430 2.0 Array Annotation Data (mouse4302). In, R package version 1.16.0. Edition. 22 Page 22 of 58 Lu W, Yamamoto V, Ortega B, Baltimore D (2004) Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth. Cell 119:97-108.

Meyer-Franke A, Kaplan MR, Pfrieger FW, Barres BA (1995) Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron 15:805-819.

Oya T, Zhao YL, Takagawa K, Kawaguchi M, Shirakawa K, Yamauchi T, Sasahara M (2002) Platelet- derived growth factor-b expression induced after rat peripheral nerve injuries. Glia 38:303-312.

Ranscht B, Clapshaw PA, Price J, Noble M, Seifert W (1982) Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc Natl Acad Sci U S A 79:2709-2713.

Schaeren-Wiemers N, Gerfin-Moser A (1993) A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100:431-440.

Segal MM, Baughman RW, Jones KA, Huettner JE (1998) Mass cultures and microislands of neurons from postnatal rat brain. In: Culturing Nerve Cells, 2nd Edition (Banker G, Goslin K, eds), pp 309 - 338. Cambridge, Massachusetts: The MIT Press.

Silvestre JS, Thery C, Hamard G, Boddaert J, Aguilar B, Delcayre A, Houbron C, Tamarat R, Blanc- Brude O, Heeneman S, Clergue M, Duriez M, Merval R, Levy B, Tedgui A, Amigorena S, Mallat Z (2005) Lactadherin promotes VEGF-dependent neovascularization. Nat Med 11:499- 506.

Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98:5116-5121.

Xing Y, Kapur K, Wong WH (2006) Probe selection and expression index computation of affymetrix exon arrays. PLoS ONE 1:e88.

Zuo Y, Lubischer JL, Kang H, Tian L, Mikesh M, Marks A, Scofield VL, Maika S, Newman C, Krieg P, Thompson WJ (2004) Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination. J Neurosci 24:10999- 11009.

23 Page 23 of 58 A Wild Type B S100β-EGFP C Single Sort D Double Sort 97.7% 99.8% 150 150

100 150 s s s s 80 l l l l l l l 100 l 100 e e e 0.01% e 35.3% C C C C

100 f f f f o o o o

60 r r r r e e e e b b b b m m m m u u u u 40 N

50 N N N 50 50

20

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 EGFP Fluorescence EGFP Fluorescence EGFP Fluorescence EGFP Fluorescence

Supplemental Figure S1

Page 24 of 58 Page 25 of 58 Page 26 of 58 Page 27 of 58 Page 28 of 58 Page 29 of 58 Page 30 of 58 EGFP Negative Oligodendrocytes 104 7.04

103

20.3 102 EGFP Positive Astrocytes 101 OLs (O4 antibody labeling)

100 100 101 102 103 104 EGFP Fluorescence

Supplemental Figure S8

Page 31 of 58 Page 32 of 58 Page 33 of 58 Page 34 of 58 Supplemental Table S1: Gene expression levels for well-described markers of astrocytes, oligodendrocytes, neurons, microglia, and endothelial cells.

(A) Well described astrocyte markers Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Astrocytes Neurons Neurons OPCs OLs Myelin Neurons Neurons Forebrain Gene Gene Title Astroglia P1 P7 P17 Gray P17 P7n P16n OLs P7 P16 P17 Symbol

1451627_a_at 436 552 3500 9010 10273 82 224 127 92978211 601 Glt-1 Slc1a2 (solute carrier family 1, member 2), glutamate transporter EAAT2 1448397_at 276 125 1639 17089 23031 30 159 73 63 112 514 556 3310 Cx30 Gjb6 ( protein beta 6); 30 1440142_s_at 16201 15463 9742 13827 4710 16 31 13 47 77 27 63 1257 Gfap Glial fibrillary acidic protein 1452031_at 4380 25844 24681 21057 25989 4 216 2274 421 240 283 190 3857 Glast Slc1a3 (solute carrier family 1, member 3), glutamate transporter EAAT1 1434449_at 13089 22366 22900 24431 20901 44 195 192 165 345 73 238 4975 Aqp4 4 1424714_at 10450 3659 5205 13855 14011 5 10 135 83 89 13 38 1206 Aldoc aldolase 3, C isoform (Zebrin II) 1421841_at 2925 9511 12169 18684 17783 269 376 569 406 196 141 285 1916 Fgfr3 fibroblast growth factor 3

(B) Well described OPC and oligodendrocyte markers Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Astrocytes Neurons Neurons OPCs OLs Myelin Neurons Neurons Forebrain Gene Gene Title Astroglia P1 P7 P17 Gray P17 P7n P16n OLs P7 P16 P17 Symbol

1423341_at 77 329 399 445 55 35 6 13911 2950 40 257 884 347 NG2 Cspg4 (chondroitin sulfate proteoglycan 4) 1451689_a_at 32 27 153 222 39 20 55 13568 9546 10024 19 25 516 Sox10 SRY-box containing gene 10 1421917_at 485 277 795 1257 26 20 12 24131 13069 36 442 319 2246 Pdgfra platelet derived growth factor receptor, alpha polypeptide 1451689_a_at 32 27 153 222 39 20 55 13568 9546 10024 19 25 516 Sox10 SRY-box containing gene 10 1435214_at 78 31 9 47 30 18 18 52 5559 10073 34 31 366 Cx47 Gja12 (gap junction membrane channel protein alpha 12); Connexin 47 1451961_a_at 74 109 72 113 31 15 150 91 21113 30547 36 127 1405 Mbp Myelin basic protein 1448768_at 12 2 7 14 3 3 6 14 5983 24964 2 2 1451 Mog Myelin oligodendrocyte glycoprotein; MOG antibody 1419064_a_at 6 5512297 4 17265 1615 4570 12 25 324 Ugt8a UDP galactosyltransferase 8A; GalC antibody 1454078_a_at 84 102 99 126 75 111 223 3997 7970 12018 168 142 556 Gal3st1 galactose-3-O-sulfotransferase 1; O4 antibody 1445656_at 16 52 25 25 21 14 40 10 5665 13336 629628 Mobp Myelin-associated oligodendrocytic basic protein 1460219_at 43 17 103 48 57 55 119 84 18698 27147 37 74 3456 Mag Myelin-associated glycoprotein 1432558_a_at 171 37 81 57 121 18 55 5 3473 29320 46 301 1067 Mal Myelin and lymphocyte protein, T-cell differentiation protein

(C) Well described neuron markers Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Astrocytes Neurons Neurons OPCs OLs Myelin Neurons Neurons Forebrain Gene Gene Title Astroglia P1 P7 P17 Gray P17 P7n P16n OLs P7 P16 P17 Symbol

1426255_at 41 100 169 109 112 6377 8976 351 190 247 7599 6793 9173 Nefl , light polypeptide 1436889_at 7 16 41 69 50 3915 10357 670 224 37 2660 6679 9692 Gabra1 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 1 1433884_at 315 306 326 339 197 13271 18197 749 416 239 15721 17841 18508 Syt1 Synaptotagmin I 1451674_at 201 305 234 283 204 17213 16949 1537 311 163 13915 16038 16782 KCC2 Slc12a5 (solute carrier family 12, member 5); K-/Cl+ cotransporter 2 1416828_at 36 249 295 344 227 17172 21916 2341 518 159 13438 17725 23064 Snap25 Synaptosomal-associated protein 25 1451595_a_at 11 28 45 13 6 4613 3350 1100 302 32 3791 2894 2922 Kcnq2 Potassium voltage-gated channel, subfamily Q, member 2 1434800_at 39 115 152 300 111 5976 7399 113 64 45 2643 6132 6277 Sv2b synaptic vesicle glycoprotein 2 b

(D) Well described microglia markers Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Astrocytes Neurons Neurons OPCs OLs Myelin Neurons Neurons Microglia- Gene Gene Title Astroglia P1 P7 P17 Gray P17 P7n P16n OLs P7 P16 OPCs* Symbol

1449164_at 226 99 81 52 74 28 134 412 90 106 63 116 5514 Cd68 CD68 antigen, macrosialin, Scard1 1422124_a_at 43 56 84 36 25 61 105 287 68 59 36 67 6068 Ptprc Protein tyrosine phosphatase, receptor type, C; CD45 1419607_at 22 63 45 67 31 14 72 197 44 40 19 24 5437 Tnf Tumor necrosis factor *Mixed preparation of microglia-OPCs containing ~1/3 microglia

(E) Well described endothelial cell markers Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Astrocytes Neurons Neurons OPCs OLs Myelin Neurons Neurons Endothelial Gene Gene Title Astroglia P1 P7 P17 Gray P17 P7n P16n OLs P7 P16 Cells Symbol

1448873_at 188 94 40 78 47 67 87 42 43 12 227 1462 7586 Ocln Occludin 1435386_at 12 18 76 141 33 31 48 334 481 69 751 3295 10694 Vwf Von Willebrand factor homolog 1418788_at 24 11 44 84 59 32 101 28 4 6 345 3411 12080 Tie2 Tek (endothelial-specific receptor tyrosine kinase) 1417839_at 34 55 410 488 284 54 384 78 38 28 4303 16956 31524 Cldn5 Claudin 5 1421287_a_at 10 9 10 8 21 32 33 4 6 8 51 429 1524 Pecam1 Platelet/endothelial cell adhesion molecule 1

(F) S100 Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Astrocytes Neurons Neurons OPCs OLs Myelin Neurons Neurons Forebrain Gene Gene Title Astroglia P1 P7 P17 Gray P17 P7n P16n OLs P7 P16 P17 Symbol

1434342_at 345 330 2136 9066 12426 24 204 3594 9536 10802 56 101 3388 S100b , beta polypeptide, neural

Page 35 of 58 Supplemental Table S2 GeneChip file name, age, sample type, and sample name and grouping for statistical analysis by SAM. A B C D E CEL FILE NAME AGE SAMPLE TYPE SAMPLE NAME A O N AY OPC GC A 3’-Array Samples postnatal day vs. vs. vs. vs. vs. vs. vs. X X X AO MOG-O MOG-O Cult 1 Ben_Emery_GC_b 16 (C57BL/6) GC OLs X O X ------GC ---- 2 Ben_Emery_MOG_b 16 (C57BL/6) MOG Myelin OLs X O X ---- MOG-O MOG-O ---- 3 Ben_GC_a3 16 (C57BL/6) GC OLs X O X ------GC ---- 4 Ben_GC_c 16 (C57BL/6) GC OLs X O X ------GC ---- 5 Ben_MOG_a2 16 (C57BL/6) MOG Myelin OLs X O X ---- MOG-O MOG-O ---- 6 Ben_MOG_c 16 (C57BL/6) MOG Myelin OLs X O X ---- MOG-O MOG-O ---- 7 Ben_PDGF_c 16 (C57BL/6) PDGFR OPCs ------OPC ------8 Cahoy_10d_neg1_p16 16 Negative Neurons P16 X X ------9 Cahoy_11d_neg1_P16 16 Neg (Bsl1) Neurons P16n X X N ------10 Cahoy_5a_pos 7 Positive Astros P7 A X X AY ------11 Cahoy_5b_neg 7 Negative Neurons P7 X X ------12 Cahoy_6a_pos 7 Positive Astros P7 A X X AY ------13 Cahoy_6b_neg 7 Negative Neurons P7 X X ------14 Cahoy_7a_pos 8 Positive Astros P8 A X X AY ------15 Cahoy_8b_neg 7 Negative Neurons P7 X X ------16 John_13B 16 Negative Neurons P16 X X ------17 John_14C Culture d9 FACS pos Cultured FACS Pos ------18 John_14D Culture d9 FACS all Cultured FACS All ------19 John_14F Culture d9 Culture Cultured Astroglia ------Cult 20 John_18B 26 Negative Neurons P26 ------21 John_19C Culture d12 FACS pos Cultured FACS Pos ------22 John_19D Culture d12 FACS all Cultured FACS All ------23 John_19E Culture d12 Culture Cultured Astroglia ------Cult 24 John_22A P17 17 Positive Astros P17 A X X AO ------AO 25 John_23E Cult Culture d33 Culture Cultured Astroglia ------Cult 26 John_24A P17 17 Positive Astros P17 A X X AO ------AO 27 John_25G P17G 17 Gray-single Astros P17g A X X AO ------AO 28 John_30O PDRFRa 17 PDGFR OPCs ------OPC ------29 John_30P GC 17 GC OLs X O X ------GC ---- 30 John_30Q MOG 17 MOG Myelin OLs X O X ---- MOG-O MOG-O ---- 31 John_33A-P30-pos 30 Positive Astros P30 ------32 John_34A-P1-pos 1 Positive Astros P1 ------33 John_35H-P17cortex 17 Forebrain Forebrain P17 ------34 John_38B NegB P7 7 Neg (Bsl1) Neurons P7n X X N ------35 John_41FCS Cult Culture d28 Culture Cultured Astroglia ------Cult 36 John_41NB Cult Culture d28 Cult: serum-free RGCm 8d Astroglia ------37 John_jlz-5-36G-P17 17 Gray Astros P17g A X X AO ------AO 38 Lu_32A 17 Positive Astros P17 A X X AO ------AO Exon Array Sample Information Exon-Array Samples Labeling Same Biological Protocol Sample As 3’-Array 39 John_56A_MoEx-1_0-st 7 Positive Astros P7 A X X 100 ng labeling Same as Cahoy_6a 40 John_61D_MoEx-1_0-st 16 Neg (Bsl1) Neurons P16n X X N 100 ng labeling Same as Cahoy_11d 41 John_69E_MoEx-1_0-st Culture d12 Culture Cultured Astroglia ------100 ng labeling Same as John_19E 42 John_72A_MoEx-1_0-st 17 Positive Astros P17 A X X 100 ng labeling Same as John_22A 43 John_79E_MoEx-1_0-st Culture d12 Culture Cultured Astroglia ------1 μg labeling Same as John_19E 44 John_80O_MoEx-1_0-st 17 PDGFR OPCs ------1 μg labeling Same as John_30O 45 John_80P_MoEx-1_0-st 17 GC OLs X O X 1 μg labeling Same as John_30P 46 John_80Q_MoEx-1_0-st 17 MOG Myelin OLs X O X 1 μg labeling Same as John_30Q 47 John_81A_MoEx-1_0-st 7 Positive Astros P7 A X X 1 μg labeling Not run on 3’-arrays 48 John_81B_MoEx-1_0-st 7 Negative Neurons P7 X X N 1 μg labeling Not run on 3’-arrays Page 36 of 58 Supplemental Table S3a Example Page: 3'-Array MAS 5.0 master data table of 20,932 probe sets with significant expression in the CNS.

Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Astrocytes Neurons Neurons Neurons Neurons OPCs Oligos Myelin Forebrain Gene Symbol Gene Title Astroglia P1 P7-P8 P17 Gray P17 P7n P7 P16n P16 Oligos P17

1426508_at 6209 525 5652 3710 756 70 67 69 74 64 76 110 208 Gfap glial fibrillary acidic protein 1426509_s_at 5958 413 3988 3541 681 6 13 3 29 25 16 31 83 Gfap glial fibrillary acidic protein 1440142_s_at 16201 15463 9742 13827 4710 16 27 31 63 13 47 77 1257 Gfap glial fibrillary acidic protein 1426995_a_at 737 476 738 501 492 366 531 388 377 670 700 816 412 Gfer growth factor, erv1 (S. cerevisiae)-like (augmenter of liv 1452272_a_at 749 418 518 385 418 335 393 365 319 496 441 513 360 Gfer growth factor, erv1 (S. cerevisiae)-like (augmenter of liv 1416570_s_at 1373 857 1149 1398 1229 558 642 928 981 835 1078 887 849 Gfm1 G elongation factor, mitochondrial 1 1448381_at 377 557 469 537 487 383 385 546 484 313 296 312 603 Gfm1 G elongation factor, mitochondrial 1 1426590_at 316 158 254 270 307 262 210 184 185 178 191 164 189 Gfm2 G elongation factor, mitochondrial 2 1442461_at 20 81 14 25 35 113 47 147 85 158 82 291 121 Gfod1 glucose-fructose oxidoreductase domain containing 1 1455599_at 15 78 86 97 128 752 564 959 638 605 431 392 1310 Gfod1 glucose-fructose oxidoreductase domain containing 1 1451429_at 784 542 486 403 529 860 1178 891 663 451 192 172 639 Gfod2 glucose-fructose oxidoreductase domain containing 2 1418753_at 805 385 393 464 450 296 299 331 253 650 368 410 320 Gfpt2 glutamine fructose-6-phosphate transaminase 2 1439015_at 975 537 1597 3551 2881 495 711 773 605 5882 2972 2622 1042 Gfra1 glial cell line derived neurotrophic factor family recepto 1450440_at 121 11 135 224 250 48 63 38 94 580 330 407 83 Gfra1 glial cell line derived neurotrophic factor family recepto 1433716_x_at 103 94 104 101 37 1598 1142 2302 2077 1495 194 12 1718 Gfra2 glial cell line derived neurotrophic factor family recepto 1459847_x_at 89 140 68 68 65 491 289 719 507 319 104 21 414 Gfra2 glial cell line derived neurotrophic factor family recepto 1450835_a_at 95 98 69 50 92 228 337 493 422 194 124 112 260 Gfra4 glial cell line derived neurotrophic factor family recepto 1423834_s_at 298 46 184 288 129 354 601 552 607 674 417 373 52 Gga1 golgi associated, gamma adaptin ear containing, ARF b 1428141_at 739 386 385 369 353 1862 1152 1187 1186 933 416 144 718 Gga2 golgi associated, gamma adaptin ear containing, ARF b 1438008_at 248 313 364 461 459 655 827 783 766 1214 779 517 563 Gga3 golgi associated, gamma adaptin ear containing, ARF b 1419595_a_at 2088 1985 3819 3333 2629 472 845 616 538 3387 2170 2177 543 Ggh /// LOC6gamma-glutamyl hydrolase /// similar to Gamma-glutam 1433904_at 72 109 178 272 352 78 29 37 47 96 94 113 30 Ggnbp1 gametogenetin binding protein 1 1426011_a_at 1965 2112 1672 1717 2395 2888 1942 2409 2143 1681 1487 1802 1896 Ggnbp2 gametogenetin binding protein 2 1419505_a_at 202 144 197 170 208 207 229 264 216 207 207 232 199 Ggps1 geranylgeranyl diphosphate synthase 1 1419506_at 544 750 289 385 526 542 392 418 479 549 446 501 563 Ggps1 geranylgeranyl diphosphate synthase 1 1419805_s_at 296 209 264 197 182 285 388 331 281 287 364 489 299 Ggps1 geranylgeranyl diphosphate synthase 1 1429356_s_at 151 325 149 191 265 212 198 298 190 177 150 141 311 Ggps1 geranylgeranyl diphosphate synthase 1 1448485_at 103 36 113 296 217 75 19 63 286 55 151 223 72 Ggt1 gamma-glutamyltransferase 1 1418483_a_at 4791 803 953 534 229 70 179 30 590 72 27 36 134 Ggta1 glycoprotein galactosyltransferase alpha 1, 3 1451094_at 240 130 247 376 347 530 1179 1289 975 986 804 699 346 Ggtl3 gamma-glutamyltransferase-like 3 1418216_at 65 36 18 32 14 106 129 18 416 30 17 13 81 Ggtla1 gamma-glutamyltransferase-like activity 1 1415881_at 6660 2925 5324 6829 8444 2245 2611 5687 5313 1972 2899 4171 7111 Ghitm growth hormone inducible transmembrane protein 1415882_at 5667 1933 3104 3834 3956 2943 2570 3462 3832 2322 2797 2542 3901 Ghitm growth hormone inducible transmembrane protein 1417962_s_at 3156 1001 1062 2374 2595 206 443 303 858 725 186 36 574 Ghr growth 1451501_a_at 610 296 433 640 877 616180 278 179 68 35 105 Ghr growth hormone receptor 1417848_at 366 1935 990 567 198 3576 2259 914 882 2522 1150 925 1594 Gig1 glucocorticoid induced gene 1 1417849_at 57 622 385 240 25 402 1077 231 432 1095 318 338 167 Gig1 glucocorticoid induced gene 1 1449988_at 13 18 28 18 9 13 69 4 354 347118 Gimap1 GTPase, IMAP family member 1 1424375_s_at 386 234259333936 249 27 28 34 49 Gimap4 GTPase, IMAP family member 4 1427891_at 7 9 16 19 8 10 222 76 992 19 5 21 81 Gimap6 GTPase, IMAP family member 6 1456061_at 26 48 28 15 28 65 118 44 822 52 37 31 124 Gimap8 GTPase, IMAP family member 8 1452598_at 235 528 344 122 36 153 170 83 78 379 138 94 63 Gins4 GINS complex subunit 4 (Sld5 homolog) 1422763_at 1049 532 1314 800 681 681 1222 1409 1248 961 4193 3107 973 Gipc1 GIPC PDZ domain containing family, member 1 1454759_at 344 187 451 511 288 566 834 700 912 443 425 892 249 Git1 G protein-coupled receptor kinase-interactor 1 1423391_at 1035 777 723 734 625 611 753 497 1180 1296 1075 1033 585 Git2 G protein-coupled receptor kinase-interactor 2 1435925_at 290 238 204 285 261 154 231 264 460 502 339 344 205 Git2 G protein-coupled receptor kinase-interactor 2 1415800_at 8002 20405 23676 27241 29617 316 1546 572 2677 134 126 319 9602 Gja1 gap junction membrane channel protein alpha 1 1415801_at 179 321 1279 1394 1844 7 6728448 8 10186 Gja1 gap junction membrane channel protein alpha 1 1437992_x_at 11386 27557 22812 30532 28898 748 3185 1049 4604 241 334 585 15984 Gja1 gap junction membrane channel protein alpha 1 1438650_x_at 3954 18516 19897 10691 23824 230 694 413 1515 75 105 208 9801 Gja1 gap junction membrane channel protein alpha 1 1438945_x_at 5558 17781 16555 15313 19892 230 1061 585 2128 65 116 196 9399 Gja1 gap junction membrane channel protein alpha 1 1438973_x_at 363 899 382 419 370 167 191 154 251 72 57 127 776 Gja1 gap junction membrane channel protein alpha 1 1435214_at 78 31 9 47 30 18 34 18 31 52 5559 10073 366 Gja12 gap junction membrane channel protein alpha 12 1450483_at 33445210256201956 3533 105 Gja12 gap junction membrane channel protein alpha 12 1416255_at 32 58 119 237 78 127 443 66 1139 45 19 27 181 Gja4 gap junction membrane channel protein alpha 4 1447787_x_at 17 287 178 75 5 177 178 6 536 54719Gja7 gap junction membrane channel protein alpha 7 1449094_at 274 1695 746 298 189 874 885 188 1911 31 22 20 414 Gja7 gap junction membrane channel protein alpha 7 1435297_at 19 63 43 50 31 836 1250 696 681 126 48 27 493 Gja9 gap junction membrane channel protein alpha 9 1448766_at 65567375591 77 773 24 Gjb1 gap junction membrane channel protein beta 1 1448767_s_at 8 5 6 16 9 15 7 46 9 262 260 2537 25 Gjb1 gap junction membrane channel protein beta 1 1423271_at 39 181 390 1329 845 60 550 139 380 60 32 52 233 Gjb2 gap junction membrane channel protein beta 2 1448397_at 276 125 1639 17089 23031 30 514 159 556 73 63 112 3310 Gjb6 gap junction membrane channel protein beta 6 1422042_at 107 16126033124 192651133 1152 147 Gje1 gap junction membrane channel protein epsilon 1 1445523_at 8 221412518 35 58 29 63 200 304 4 Gje1 Gap junction membrane channel protein epsilon 1

Page 37 of 58 Supplemental Table S3b Example Page: 3'-Array dChip master data table of 20,932 probe sets with expression in the CNS. Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Astrocytes Neurons Neurons Neurons Neurons OPCs Oligos Myelin Forebrain Gene Symbol Astroglia P1 P7-P8 P17 Gray P17 P7n P7 P16n P16 Oligos P17 1426508_at 11.08 8.03 11.02 10.48 8.51 6.55 6.46 6.98 6.71 6.29 6.54 6.86 7.08 Gfap 1426509_s_at 11.70 9.95 11.65 11.61 9.90 5.57 5.58 6.10 6.26 5.76 6.31 7.10 7.52 Gfap 1440142_s_at 12.35 12.05 11.87 12.00 10.76 4.63 5.11 4.93 5.56 4.62 5.23 5.78 9.05 Gfap 1426995_a_at 9.09 8.47 8.85 8.81 8.71 8.53 8.68 8.36 8.47 8.81 8.74 8.98 8.38 Gfer 1452272_a_at 9.12 8.94 8.59 8.40 8.33 8.05 8.27 8.00 7.99 8.57 8.73 8.71 8.20 Gfer 1416570_s_at 9.45 9.03 9.24 9.36 9.32 8.33 8.49 8.84 9.03 8.91 9.13 9.04 9.06 Gfm1 1448381_at 8.42 8.98 8.73 8.79 8.65 8.36 8.39 8.50 8.57 8.13 8.21 8.18 8.86 Gfm1 1426590_at 7.74 7.02 7.54 7.67 7.63 7.58 7.32 7.39 7.31 7.30 7.32 7.20 7.33 Gfm2 1442461_at 6.48 7.38 6.32 6.37 6.90 7.31 6.53 6.90 6.50 6.82 6.96 7.43 7.21 Gfod1 1455599_at 8.58 9.15 8.33 8.89 9.21 10.00 9.31 9.67 9.64 9.45 9.13 9.33 10.50 Gfod1 1451429_at 9.04 8.01 8.50 8.30 8.13 9.28 9.45 9.04 9.02 8.35 7.44 7.40 8.52 Gfod2 1418753_at 9.36 8.91 8.61 9.02 9.14 8.87 8.32 8.47 8.15 8.88 8.47 8.39 8.87 Gfpt2 1439015_at 9.31 8.74 9.61 10.50 10.33 8.56 8.97 9.16 9.16 11.40 10.48 10.44 9.18 Gfra1 1450440_at 6.83 6.50 6.94 7.60 7.58 6.80 6.56 6.63 6.69 8.44 7.96 8.19 7.03 Gfra1 1433716_x_at 5.57 5.65 5.65 5.46 4.81 9.45 8.73 9.77 9.54 9.06 6.48 3.76 9.40 Gfra2 1459847_x_at 5.89 5.63 5.72 5.45 5.13 8.83 8.30 9.10 9.03 8.63 6.35 4.80 8.73 Gfra2 1450835_a_at 8.17 8.43 7.71 7.82 8.34 8.32 8.46 9.19 9.01 8.02 8.04 7.96 8.81 Gfra4 1423834_s_at 8.31 8.33 8.43 8.62 8.55 8.40 8.46 8.20 8.30 8.79 8.52 8.56 8.17 Gga1 1428141_at 9.48 8.58 8.68 8.74 8.69 10.46 9.92 9.76 9.85 9.59 8.81 7.67 9.43 Gga2 1438008_at 7.81 8.14 8.30 8.42 8.34 9.34 9.24 9.20 9.17 9.78 9.31 8.74 9.09 Gga3 1419595_a_at 10.10 10.11 10.91 10.87 10.49 8.55 9.08 8.31 8.59 10.96 10.16 10.19 8.63 Ggh 1433904_at 7.19 7.98 8.18 8.45 8.72 7.23 7.03 7.23 6.88 7.34 6.97 7.00 7.19 Ggnbp1 1426011_a_at 9.90 9.97 9.76 9.65 9.89 10.28 9.77 9.90 9.95 9.57 9.55 9.69 9.81 Ggnbp2 1419505_a_at 7.13 6.61 7.00 6.75 7.08 7.17 7.22 7.04 7.10 6.89 7.12 7.63 7.19 Ggps1 1419506_at 8.38 8.68 7.57 8.48 8.58 8.29 7.89 7.52 8.01 8.22 8.26 8.27 8.28 Ggps1 1419805_s_at 7.51 6.80 7.11 6.50 6.77 7.32 7.86 7.28 7.39 7.50 7.76 8.43 7.24 Ggps1 1429356_s_at 7.08 7.67 7.25 7.46 7.33 7.49 7.41 7.07 7.25 7.22 7.10 6.88 7.51 Ggps1 1448485_at 6.82 6.14 6.80 7.68 7.48 5.87 6.16 5.97 7.48 6.25 7.03 7.48 6.14 Ggt1 1418483_a_at 11.25 9.33 9.18 8.80 7.19 6.33 7.07 5.55 8.63 6.11 5.45 5.69 6.96 Ggta1 1451094_at 8.01 7.81 7.90 8.38 8.55 8.67 9.31 9.39 9.13 9.26 8.93 8.92 8.61 Ggtl3 1418216_at 6.92 6.59 6.64 6.69 6.68 5.96 6.77 6.64 7.81 6.00 6.08 6.42 6.23 Ggtla1 1419502_at 8.58 7.47 8.86 8.78 8.68 6.60 7.43 6.70 7.67 8.96 8.47 8.40 7.03 Ghdc 1415881_at 11.42 10.43 11.26 11.50 11.65 10.47 10.48 11.30 11.37 9.94 10.45 10.83 11.68 Ghitm 1415882_at 11.83 10.97 11.35 11.65 11.72 11.21 11.00 11.35 11.62 11.04 11.04 10.94 11.68 Ghitm 1417962_s_at 10.19 8.70 9.04 9.85 10.03 7.10 7.95 6.91 8.66 8.40 6.54 5.18 8.12 Ghr 1451501_a_at 8.55 7.41 8.09 8.60 8.86 6.27 6.62 6.07 7.45 6.93 6.04 5.77 6.89 Ghr 1449988_at 3.86 3.83 4.61 4.63 4.23 4.28 6.30 4.54 8.72 4.15 4.14 4.78 6.09 Gimap1 1424375_s_at 6.00 7.51 6.33 6.42 6.86 6.85 6.37 7.25 7.16 6.12 6.40 6.44 7.19 Gimap4 1427891_at 4.80 5.00 5.23 5.43 5.56 5.17 7.57 5.55 9.76 4.97 5.00 5.21 6.55 Gimap6 1456061_at 5.88 5.71 5.27 5.68 5.63 5.94 6.59 5.70 8.71 5.44 5.62 5.52 6.58 Gimap8 1452598_at 7.61 8.27 7.78 6.81 6.64 6.92 7.09 6.52 6.47 8.02 6.90 6.57 6.73 Gins1 1417913_at 9.54 8.42 9.06 9.14 8.87 8.34 8.51 8.43 8.70 9.32 9.62 10.27 8.65 Gins4 1422763_at 9.03 8.39 9.36 9.00 8.58 8.70 9.26 9.17 9.32 9.18 10.92 10.50 9.08 Gipc1 1454759_at 9.57 9.88 9.67 9.54 9.28 10.42 10.38 10.06 10.34 9.49 9.97 10.82 10.49 Git1 1423391_at 8.96 8.66 8.52 8.63 8.45 8.46 8.46 7.99 9.12 9.29 9.11 9.09 8.51 Git2 1435925_at 7.80 7.44 7.63 7.78 7.54 7.23 7.49 7.14 8.46 8.40 8.00 8.13 7.49 Git2 1415800_at 11.72 12.67 13.19 13.26 13.41 7.61 9.68 8.03 10.42 6.49 6.71 7.82 11.97 Gja1 1415801_at 7.14 7.30 9.37 9.65 9.80 5.48 6.01 5.62 5.89 5.53 5.66 5.92 6.92 Gja1 1437992_x_at 12.03 13.00 13.15 13.28 13.36 8.01 10.11 8.51 10.89 6.89 7.22 8.10 12.47 Gja1 1438650_x_at 11.81 12.69 12.88 13.17 13.22 7.36 9.82 8.21 10.73 6.88 7.03 8.03 12.15 Gja1 1438945_x_at 11.46 12.44 12.77 12.71 12.88 7.19 9.51 7.99 10.27 6.37 6.64 7.58 11.85 Gja1 1438973_x_at 5.85 6.36 5.91 5.90 6.25 5.62 5.30 5.41 5.22 5.17 5.23 5.58 6.24 Gja1 1435214_at 7.63 7.92 7.35 7.32 7.63 7.96 7.46 7.79 7.47 7.19 11.31 12.01 8.53 Gja12 1450483_at 5.45 5.44 5.41 5.32 5.53 5.27 5.41 5.71 5.37 5.08 10.03 10.76 6.74 Gja12 1416255_at 5.74 5.03 6.24 7.35 5.72 6.29 7.92 5.93 9.29 4.92 4.87 5.11 6.48 Gja4 1440999_at 8.84 9.26 8.17 7.51 7.01 10.05 9.19 8.54 8.51 7.86 7.08 6.24 9.22 Gja5 1447787_x_at 6.02 7.72 7.33 6.78 5.77 7.86 7.50 5.61 8.26 5.28 5.82 6.25 6.54 Gja7 1449094_at 7.49 10.05 8.88 7.85 6.73 9.16 8.93 6.70 10.06 5.38 5.08 5.21 8.07 Gja7 1435297_at 5.16 5.33 5.34 5.14 5.29 9.18 9.40 8.82 8.71 6.59 5.23 5.15 8.56 Gja9 1448766_at 5.14 5.62 5.27 5.44 5.50 5.56 5.23 4.67 4.87 6.19 5.98 8.73 6.08 Gjb1 1448767_s_at 5.65 5.15 5.53 5.50 5.52 5.44 5.50 5.79 5.57 7.62 7.89 10.72 6.55 Gjb1 1423271_at 6.19 7.07 7.98 9.72 9.10 5.89 8.42 6.16 7.91 5.64 5.88 6.21 7.42 Gjb2 1448397_at 7.31 6.89 9.43 12.59 12.98 5.77 7.87 6.44 7.96 6.32 6.21 6.85 10.22 Gjb6 1422042_at 4.85 4.99 5.09 5.16 5.56 4.87 5.02 4.89 4.81 6.95 9.14 9.06 6.09 Gje1 1445523_at 8.13 7.94 7.34 7.42 7.81 7.97 7.36 8.65 7.89 7.67 8.11 8.19 8.10 Gje1 1436210_at 6.30 7.07 7.31 7.71 7.66 7.52 7.06 6.18 6.45 7.10 6.58 7.85 6.63 Gk5 1417594_at 9.69 9.81 10.03 9.93 9.63 8.49 8.83 7.92 8.46 9.75 10.40 11.03 8.49 Gkap1 1418248_at 7.55 6.90 7.57 6.64 6.83 6.97 7.20 6.91 6.99 7.03 7.20 7.61 6.79 Gla 1416205_at 8.97 8.35 9.08 8.97 8.91 8.48 9.36 9.05 9.05 9.62 8.97 8.29 8.52 Glb1 1430385_a_at 7.73 7.56 7.82 7.66 7.84 6.77 6.35 6.47 6.39 6.24 6.19 6.10 6.68 Glb1l 1426218_at 7.77 8.89 8.28 8.52 8.85 9.52 8.74 8.77 8.81 9.41 9.07 8.97 9.48 Glcci1 1428603_at 9.59 8.71 8.78 9.23 9.22 10.80 9.88 10.37 10.11 9.27 8.86 8.79 10.26 Glcci1 1438295_at 5.77 7.76 6.49 6.01 6.27 9.02 8.13 7.10 6.88 8.17 7.53 6.75 7.08 Glcci1 1451972_at 6.99 7.23 7.05 7.48 7.56 8.17 7.42 7.27 7.59 8.62 8.10 8.11 7.56 Glcci1 1457061_at 6.18 6.35 6.67 6.62 6.29 7.35 7.68 7.77 7.84 6.28 6.30 6.70 8.03 Glcci1 1428374_at 8.32 7.17 7.17 6.97 6.65 8.87 8.80 8.98 8.96 9.61 8.30 7.03 8.84 Glce 1416049_at 11.07 11.48 11.39 12.09 11.90 6.95 6.95 6.93 6.68 9.64 8.31 6.76 8.95 Gldc 1426344_at 9.45 9.41 9.37 9.26 9.30 10.54 9.89 9.52 9.58 9.39 9.29 9.25 9.72 Gle1l 1452033_at 8.33 8.70 8.25 8.10 8.43 9.02 8.62 9.12 8.77 8.16 8.85 9.02 9.04 Gle1l 1417087_at 9.86 10.84 10.27 10.48 10.45 9.90 9.84 9.94 10.33 10.68 10.36 10.29 10.48 Glg1 1448579_at 10.79 11.38 11.46 11.60 11.36 10.46 11.15 11.24 11.54 11.79 11.23 11.23 11.23 Glg1 1448580_at 9.80 10.61 10.04 9.56 9.45 9.30 9.13 8.77 9.20 9.76 9.46 9.41 9.20 Glg1 1460554_s_at 10.00 10.43 10.52 10.64 10.36 10.13 10.26 10.41 10.52 10.88 10.17 10.17 10.10 Glg1 1446086_s_at 7.87 8.14 8.43 8.34 8.38 6.87 6.65 6.55 6.37 5.99 6.15 6.21 6.66 Gli2 1459211_at 9.57 9.96 9.75 9.87 9.86 8.54 8.47 8.70 8.44 8.19 9.22 10.03 8.71 Gli2 1455154_at 9.61 10.42 10.32 10.75 10.76 5.44 6.18 5.70 6.19 5.06 5.33 5.48 7.35 Gli3 1456067_at 9.51 10.66 9.93 10.61 10.68 6.88 6.49 6.41 6.43 5.69 5.94 6.10 7.80 Gli3

Page 38 of 58 Supplemental Table S4 First Page: Genes statistically enriched in astrocytes. Probe Set ID Gene Name Fold Enriched In Astrocytes 1 1440142_s_at Gfap 84.9 2 1434449_at Aqp4 79.8 3 1430700_a_at Pla2g7 65.1 4 1436611_at Slc39a12 62.2 5 1448139_at Mlc1 58.6 6 1436231_at AU067665 57.9 7 1418937_at Dio2 56.0 8 1428114_at Slc14a1 54.4 9 1424400_a_at Aldh1l1 54.0 10 1419559_at Cyp4f14 53.5 11 1424714_at Aldoc 52.9 12 1427284_a_at Ttpa 52.1 13 1422428_at Acsbg1 50.0 14 1434201_at Chrdl1 49.1 15 1436115_at Gm266 48.6 16 1434096_at Slc4a4 46.9 17 1451627_a_at Slc1a2 46.7 18 1428964_at Slc25a18 45.6 19 1426340_at Slc1a3 43.0 20 1417408_at F3 40.6 21 1428923_at Ppp1r3g 37.8 22 1449316_at Cyp4f15 37.1 23 1428303_at 1500005I02Rik 36.7 24 1418533_s_at Fzd2 35.5 25 1457800_at 2900019G14Rik 35.2 26 1422869_at Mertk 33.0 27 1450850_at Vil2 32.8 28 1424652_at Tmem166 32.7 29 1448397_at Gjb6 32.7 30 1438020_at Hapln1 32.7 31 1436931_at Rfx4 32.7 32 1434510_at Papss2 32.7 33 1417600_at Slc15a2 32.6 34 1433691_at Ppp1r3c 32.4 35 1422782_s_at Tlr3 31.5 36 1429267_at Acot11 30.9 37 1452308_a_at Atp1a2 29.8 38 1437312_at Bmpr1b 29.7 39 1436569_at C030045D06Rik 29.4 40 1417629_at Prodh 29.4 41 1455154_at Gli3 29.3 42 1449885_at Tmem47 29.3 43 1438115_a_at Slc9a3r1 29.1 44 1426243_at Cth 28.8 45 1417151_a_at Ntsr2 28.1 46 1421093_at Slc7a10 27.5 47 1448162_at Vcam1 27.3 48 1421841_at Fgfr3 27.2 49 1424186_at Ccdc80 26.2 50 1418259_a_at Entpd2 25.7 51 1460604_at Cybrd1 25.7 52 1416640_at Kcne1l 25.6 53 1433653_at BC029169 25.4 54 1435417_at AI464131 25.3 55 1438132_at EG328479 25.2 56 1416342_at Tnc 24.8 57 1452132_at Tlcd1 24.0 58 1423571_at Edg1 23.8 59 1423844_s_at Cbs 23.4 60 1451132_at Pbxip1 23.4 61 1449245_at Grin2c 23.3 62 1439894_at A730056I06Rik 23.0 63 1424393_s_at Adhfe1 22.6 64 1423396_at Agt 22.0 65 1416049_at Gldc 21.2 66 1436555_at Slc7a2 21.2 67 1434202_a_at BC055107 20.9 68 1415800_at Gja1 20.9 69 1417273_at Pdk4 20.8 70 1435888_at Egfr 20.6 71 1424950_at Sox9 20.4 72 1426147_s_at Cldn10 20.2 73 1437030_at Plcd4 20.1 74 1423259_at Id4 19.8 75 1417429_at Fmo1 19.7 76 1433670_at Emp2 19.5 77 1436200_at Lonrf3 19.4 78 1438251_x_at Htra1 19.3 79 1415897_a_at Mgst1 19.2 80 1424737_at Thrsp 19.1

Page 39 of 58 Supplemental Table S5 First Page: Genes statistically enriched in oligodendrocytes. Probe Set ID Gene Name Fold Enriched In Oligodendrocytes 1 1448768_at Mog 211.1 2 1433785_at Mobp 199.9 3 1439785_at 9630013A20Rik 145.1 4 1442075_at AI314604 115.7 5 1419063_at Ugt8a 111.5 6 1440902_at A330104H05Rik 110.2 7 1438785_at Enpp6 92.3 8 1416003_at Cldn11 91.5 9 1441909_s_at 9530066K23Rik 88.0 10 1460219_at Mag 70.8 11 1447807_s_at Plekhh1 70.3 12 1439506_at Gm98 62.6 13 1435854_at Tmem10 59.1 14 1426960_a_at Fa2h 58.2 15 1418739_at Sgk2 56.6 16 1419529_at Il23a 55.6 17 1434094_at 6330530A05Rik 53.1 18 1449365_at Edg8 51.5 19 1456228_x_at Mbp 48.8 20 1425546_a_at Trf 48.1 21 1442138_at Gpr62 45.2 22 1424567_at Tspan2 45.0 23 1417275_at Mal 44.9 24 1434606_at Erbb3 44.3 25 1455965_at Adamts4 41.6 26 1440354_at Elovl7 41.3 27 1418086_at Ppp1r14a 41.1 28 1425468_at Plp1 37.0 29 1440813_s_at Plxnb3 35.3 30 1436974_at A230069A22Rik 35.1 31 1454078_a_at Gal3st1 33.9 32 1428792_at Bcas1 33.7 33 1447806_s_at Srpk3 33.6 34 1450483_at Gja12 33.5 35 1438044_at 1700047M11Rik 33.0 36 1453009_at Cpm 32.6 37 1450241_a_at Evi2a 32.3 38 1448558_a_at Pla2g4a 31.0 39 1415812_at Gsn 25.7 40 1426044_a_at Prkcq 25.0 41 1429459_at Sema3d 24.2 42 1434399_at Galnt6 23.5 43 1423946_at Pdlim2 23.0 44 1434432_at Rffl 22.9 45 1456833_at Gpr17 22.7 46 1430755_at 4930452G13Rik 21.9 47 1420962_at Hapln2 21.8 48 1448767_s_at Gjb1 21.4 49 1435642_at Sft2d1 21.4 50 1433667_at Lgi3 20.3 51 1427420_at Nkx6-2 19.8 52 1451689_a_at Sox10 19.7 53 1417143_at Edg2 19.6 54 1449383_at Adssl1 19.2 55 1422042_at Gje1 18.5 56 1426663_s_at Slc45a3 18.4 57 1439648_at Anln 18.3 58 1450813_a_at Tnni1 18.1 59 1429284_at 8430436F23Rik 17.9 60 1448945_at Pllp 17.7 61 1417590_at Cyp27a1 17.2 62 1428599_at Kndc1 17.1 63 1435343_at Dock10 17.1 64 1435125_at BB120497 16.8 65 1447914_x_at 4833411O04Rik 16.3 66 1436345_at 5730559C18Rik 16.2 67 1435931_at E130308A19Rik 15.8 68 1428573_at Chn2 15.4 69 1421844_at Il1rap 15.4 70 1455123_at St18 15.3 71 1452980_at 2810468N07Rik 15.0 72 1457323_at C030030A07Rik 15.0 73 1435463_s_at Myo1d 14.9 74 1422493_at Cpox 14.8 75 1451210_at Ppap2c 14.7 76 1418980_a_at Cnp1 14.6 77 1453269_at Unc5b 14.6 78 1426215_at Ddc 14.6 79 1416118_at Trim59 14.4 80 1424468_s_at Phldb1 14.3

Page 40 of 58 Supplemental Table S6 First Page: Genes statistically enriched in neurons. Probe Set ID Gene Name Fold Enriched In Neurons 1 1418047_at Neurod6 92.5 2 1434098_at Glra2 79.4 3 1426851_a_at Nov 65.5 4 1455925_at Prdm8 63.4 5 1447813_x_at Sla 62.7 6 1457984_at Crh 60.1 7 1437147_at Gabrg2 59.8 8 1435513_at Htr2c 57.9 9 1438624_x_at Hs3st2 56.0 10 1427042_at Mal2 55.9 11 1453245_at 9130024F11Rik 54.9 12 1437156_at Efcbp1 50.2 13 1423281_at Stmn2 50.1 14 1433602_at Gabra5 47.4 15 1422860_at Nts 45.0 16 1439808_at A130090K04Rik 43.7 17 1436889_at Gabra1 41.5 18 1427017_at Satb2 41.5 19 1423171_at Gpr88 41.2 20 1421990_at Syt1 39.4 21 1454926_at 4930544G21Rik 38.7 22 1435749_at Gda 37.4 23 1421175_at Myt1l 37.3 24 1428379_at Slc17a6 36.7 25 1434292_at A930034L06Rik 36.4 26 1445124_at A930009L07Rik 35.6 27 1459372_at Npas4 35.2 28 1417504_at Calb1 35.2 29 1451674_at Slc12a5 33.4 30 1452380_at Epha7 32.9 31 1428664_at Vip 32.6 32 1424852_at Mef2c 32.2 33 1422256_at Sstr2 32.0 34 1457273_at Odz2 31.8 35 1447992_s_at Pcsk2 30.3 36 1416828_at Snap25 30.3 37 1450708_at Scg2 30.3 38 1456478_at Pgm2l1 30.2 39 1436821_at Plcxd3 30.0 40 1419845_at Dlx1 30.0 41 1420955_at Vsnl1 29.8 42 1415844_at Syt4 29.2 43 1421017_at Nrg3 29.1 44 1435933_at Scn2a1 29.0 45 1418619_at Icam5 28.9 46 1454768_at Kcnf1 28.7 47 1434447_at AI838057 28.5 48 1419473_a_at Cck 28.0 49 1436094_at Vgf 27.9 50 1455148_at Tmem130 27.7 51 1439843_at Camk4 27.6 52 1436455_at Asph 27.5 53 1441369_at C030017B01Rik 27.4 54 1455469_at Slc6a7 27.0 55 1459717_at Olfr1344 26.9 56 1431049_at Ica1l 26.9 57 1452298_a_at Myo5b 26.8 58 1433787_at Nell1 26.6 59 1422520_at Nefm 26.5 60 1454672_at Nefl 26.5 61 1455913_x_at Ttr 26.4 62 1423853_at 6330527O06Rik 26.3 63 1455365_at Cdh8 26.2 64 1434800_at Sv2b 26.1 65 1423537_at Gap43 25.6 66 1457156_at Trhde 25.5 67 1436450_at D11Bwg0517e 25.4 68 1448676_at Camk2b 25.3 69 1427038_at Penk1 25.3 70 1416286_at Rgs4 25.2 71 1415904_at Lpl 24.4 72 1460608_at Cacna1b 24.4 73 1455258_at Kcnc2 24.1 74 1436237_at Ttc9 24.1 75 1450435_at L1cam 24.0 76 1452966_at 9130430L19Rik 23.5 77 1441165_s_at Clstn2 23.4 78 1423172_at Napb 23.4 79 1417507_at Cyb561 23.2 80 1453282_at Cxadr 23.0

Page 41 of 58 Supplemental Table S7: Comparison of cell type enriched genes from 3'-arrays to exon-arrays.

Astrocytes enriched genes Probe Set ID Gene Symbol 3'-Array Fold Transcript Exon-Array Enriched Cluster ID Fold Enriched 1 1440142_s_at Gfap 84.9 6791641 40.5 2 1434449_at Aqp4 79.8 6863605 70.1 3 1430700_a_at Pla2g7 65.1 6850534 55.4 4 1436611_at Slc39a12 62.2 6875182 56.0 5 1448139_at Mlc1 58.6 6837773 47.1 6 1436231_at AU067665 57.9 6988819 52.2 7 1418937_at Dio2 56.0 6802727 48.2 8 1428114_at Slc14a1 54.4 6866852 100.6 9 1424400_a_at Aldh1l1 54.0 6947936 63.9 10 1419559_at Cyp4f14 53.5 6854909 66.3 11 1424714_at Aldoc 52.9 6782694 14.6 12 1427284_a_at Ttpa 52.1 6912040 76.1 13 1422428_at Acsbg1 50.0 6995762 57.8 14 1434201_at Chrdl1 49.1 7019944 56.9 15 1436115_at Gm266 48.6 not in exon array profile 16 1434096_at Slc4a4 46.9 6932240 24.3 17 1451627_a_at Slc1a2 46.7 6879569 5.7 18 1428964_at Slc25a18 45.6 not in exon array profile 19 1426340_at Slc1a3 43.0 not in exon array profile 20 1417408_at F3 40.6 6900970 32.0 21 1428923_at Ppp1r3g 37.8 not in exon array profile 22 1449316_at Cyp4f15 37.1 6849893 121.9 23 1428303_at 1500005I02Rik 36.7 6792352 19.1 24 1418533_s_at Fzd2 35.5 6784348 47.9 25 1457800_at 2900019G14Rik 35.2 6879569 5.7 26 1422869_at Mertk 33.0 6881087 36.2 27 1450850_at Vil2 32.8 6848568 26.6 28 1424652_at Tmem166 32.7 6947332 24.3 29 1448397_at Gjb6 32.7 6824940 39.8 30 1438020_at Hapln1 32.7 6808781 9.8 31 1436931_at Rfx4 32.7 6769448 22.2 32 1434510_at Papss2 32.7 6869216 46.0 33 1417600_at Slc15a2 32.6 6845444 71.0 34 1433691_at Ppp1r3c 32.4 6872878 46.5 35 1422782_s_at Tlr3 31.5 6982102 35.0 36 1429267_at Acot11 30.9 6924247 25.3 37 1452308_a_at Atp1a2 29.8 6764138 11.5 38 1437312_at Bmpr1b 29.7 6909941 26.1 39 1436569_at C030045D06Rik 29.4 not in exon array profile 40 1417629_at Prodh 29.4 6844316 38.2

Oligodendrocyte enriched genes Probe Set ID Gene Symbol 3'-Array Fold Transcript Exon-Array Enriched Cluster ID Fold Enriched 1 1448768_at Mog 211.1 6855225 106.3 2 1433785_at Mobp 199.9 6992925 344.2 3 1439785_at 9630013A20Rik 145.1 not in exon array profile 4 1442075_at AI314604 115.7 6975930 209.2 5 1419063_at Ugt8a 111.5 6909083 114.8 6 1440902_at A330104H05Rik 110.2 6886947 309.1 7 1438785_at Enpp6 92.3 6975930 209.2 8 1416003_at Cldn11 91.5 6896524 145.9 9 1441909_s_at 9530066K23Rik 88.0 6787741 211.1 10 1460219_at Mag 70.8 6966324 243.1 11 1447807_s_at Plekhh1 70.3 not in exon array profile 12 1439506_at Gm98 62.6 not in exon array profile 13 1435854_at Tmem10 59.1 6873127 362.2 14 1426960_a_at Fa2h 58.2 6985363 179.9 15 1418739_at Sgk2 56.6 6882995 52.2 16 1419529_at Il23a 55.6 6778008 24.7 17 1434094_at 6330530A05Rik 53.1 6924900 108.5 18 1449365_at Edg8 51.5 6993756 80.9 19 1456228_x_at Mbp 48.8 6862586 58.1 20 1425546_a_at Trf 48.1 6998397 85.4

Page 42 of 58 Supplemental Table S7: Comparison of cell type enriched genes from 3'-arrays to exon-arrays.

21 1442138_at Gpr62 45.2 not in exon array profile 22 1424567_at Tspan2 45.0 6900082 32.0 23 1417275_at Mal 44.9 6890710 128.3 24 1434606_at Erbb3 44.3 6778044 94.4 25 1455965_at Adamts4 41.6 6755168 71.5 26 1440354_at Elovl7 41.3 6810063 54.3 27 1418086_at Ppp1r14a 41.1 6959487 72.2 28 1425468_at Plp1 37.0 7014035 18.4 29 1440813_s_at Plxnb3 35.3 not in exon array profile 30 1436974_at A230069A22Rik 35.1 not in exon array profile 31 1454078_a_at Gal3st1 33.9 6778355 115.8 32 1428792_at Bcas1 33.7 6893382 134.6 33 1447806_s_at Srpk3 33.6 7011952 40.9 34 1450483_at Gja12 33.5 6788655 57.7 35 1438044_at 1700047M11Rik 33.0 not in exon array profile 36 1453009_at Cpm 32.6 not in exon array profile 37 1450241_a_at Evi2a 32.3 6790046 69.2 38 1448558_a_at Pla2g4a 31.0 6762944 67.5 39 1415812_at Gsn 25.7 6876380 42.4 40 1426044_a_at Prkcq 25.0 6874979 80.9

Neuron enriched genes Probe Set ID Gene Symbol 3'-Array Fold Transcript Exon-Array Enriched Cluster ID Fold Enriched 1 1418047_at Neurod6 92.5 6953866 95.3 2 1434098_at Glra2 79.4 7020724 58.8 3 1426851_a_at Nov 65.5 6830481 42.2 4 1455925_at Prdm8 63.4 6932704 16.9 5 1447813_x_at Sla 62.7 6836341 38.1 6 1457984_at Crh 60.1 6903525 3.9 7 1437147_at Gabrg2 59.8 6787525 37.9 8 1435513_at Htr2c 57.9 7014406 16.0 9 1438624_x_at Hs3st2 56.0 6963989 19.1 10 1427042_at Mal2 55.9 6830480 51.7 11 1453245_at 9130024F11Rik 54.9 6758862 42.3 12 1437156_at Efcbp1 50.2 6919895 40.6 13 1423281_at Stmn2 50.1 6895589 55.4 14 1433602_at Gabra5 47.4 6967593 35.9 15 1422860_at Nts 45.0 6776577 15.7 16 1439808_at A130090K04Rik 43.7 not in exon array profile 17 1436889_at Gabra1 41.5 6787527 22.6 18 1427017_at Satb2 41.5 6758862 42.3 19 1423171_at Gpr88 41.2 6908487 19.2 20 1421990_at Syt1 39.4 6776872 24.0 21 1454926_at 4930544G21Rik 38.7 6760251 32.5 22 1435749_at Gda 37.4 6872051 45.4 23 1421175_at Myt1l 37.3 not in exon array profile 24 1428379_at Slc17a6 36.7 6960692 56.5 25 1434292_at A930034L06Rik 36.4 6882738 12.8 26 1445124_at A930009L07Rik 35.6 not in exon array profile 27 1459372_at Npas4 35.2 6871062 50.6 28 1417504_at Calb1 35.2 6911914 16.0 29 1451674_at Slc12a5 33.4 6883127 22.2 30 1452380_at Epha7 32.9 6912371 13.9 31 1428664_at Vip 32.6 6771858 26.1 32 1424852_at Mef2c 32.2 6808609 22.5 33 1422256_at Sstr2 32.0 not in exon array profile 34 1457273_at Odz2 31.8 not in exon array profile 35 1447992_s_at Pcsk2 30.3 6881771 22.5 36 1416828_at Snap25 30.3 6881488 26.3 37 1450708_at Scg2 30.3 6759997 30.0 38 1456478_at Pgm2l1 30.2 not in exon array profile 39 1436821_at Plcxd3 30.0 6828379 26.9 40 1419845_at Dlx1 30.0 6878021 9.6

Page 43 of 58 Supplemental Table S8 Example Page: Exon-array master data table of gene expression in the CNS.

Exon Array Accession Cultured Cultured Astros Astros Astros Neurons Neurons OPCs OLs Myelin Gene Symbol: Gene Name Transcript Number Gene Astroglia Astroglia P7 P7 P17 P7 (81b) P16n (80O) (80P) OLs Cluster ID (RefSeq) (69E) (79E) (56A) (81A) (72A) (61D) (80Q) 6791641 NM_010277 14580 14695 6001 7172 4497 7033 67 49 50 145 355 Gfap: glial fibrillary acidic protein 6854384 NM_023040 11692 508 360 282 235 302 185 212 281 272 364 Gfer: growth factor, erv1 (S. cerevisiae)-like (augmenter of liver re 6940872 NM_010278 14581 39 27 25 31 47 30 48 23 31 35 Gfi1: growth factor independent 1 6885627 NM_008114 14582 16 35 46 25 22 25 31 19 15 35 Gfi1b: growth factor independent 1B 6898162 NM_138591 28030 864 724 625 674 709 415 661 443 592 649 Gfm1: G elongation factor, mitochondrial 1 6809266 NM_177266 320806 330 368 278 340 358 262 231 316 299 247 Gfm2: G elongation factor, mitochondrial 2 6984993 NM_027469 70575 573 279 472 140 186 296 696 154 81 95 Gfod2: glucose-fructose oxidoreductase domain containing 2 6947679 NM_013528 14583 667 678 811 1016 1034 1079 931 1108 2248 1526 Gfpt1: glutamine fructose-6-phosphate transaminase 1 6780767 NM_013529 14584 476 518 173 220 314 165 100 365 268 302 Gfpt2: glutamine fructose-6-phosphate transaminase 2 6874057 NM_010279 14585 1301 1193 821 987 2069 507 591 2249 2818 3546 Gfra1: glial cell line derived neurotrophic factor family receptor alp 6820113 NM_008115 14586 30 32 40 47 30 479 709 582 159 44 Gfra2: glial cell line derived neurotrophic factor family receptor alp 6864527 NM_010280 14587 5 2591 2 2447Gfra3: glial cell line derived neurotrophic factor family receptor alp 6890979 NM_020014 14588 14 60 10 16 36 749 1343 529 332 90 Gfra4: glial cell line derived neurotrophic factor family receptor alp 6996990 NM_205844 404194 29 16 73 31 75 18 17 11 17 36 Gfral: GDNF family receptor alpha like 6831847 NM_145929 106039 900 763 732 709 771 810 796 750 842 864 Gga1: golgi associated, gamma adaptin ear containing, ARF bindin 6971017 NM_028758 74105 216 224 157 151 160 423 388 336 224 138 Gga2: golgi associated, gamma adaptin ear containing, ARF bindin 6792452 NM_173048 260302 128 127 222 196 223 501 554 540 602 432 Gga3: golgi associated, gamma adaptin ear containing, ARF bindin 6946979 NM_019802 56316 887 850 636 553 752 283 333 673 550 279 Ggcx: gamma-glutamyl carboxylase 6912042 NM_010281 14590 1354 1031 1495 1165 1598 295 271 1008 998 1181 Ggh: gamma-glutamyl hydrolase 6959477 NM_182694 243897 63 50 74 42 69 28 34 42 30 43 Ggn: gametogenetin 6849476 NM_027544 70772 20 22 48 36 117 13 27 16 23 30 Ggnbp1: gametogenetin binding protein 1 6790360 NM_153144 217039 1366 432 1102 466 987 478 1315 386 414 544 Ggnbp2: gametogenetin binding protein 2 6811121 NM_010282 14593 930 751 584 554 806 598 731 533 614 698 Ggps1: geranylgeranyl diphosphate synthase 1 6768867 NM_008116 14598 72 86 55 46 126 45 39 15 79 121 Ggt1: gamma-glutamyltransferase 1 6782246 NM_027819 71522 22 17 8 18 21 14 12 7 11 23 Ggt6: gamma-glutamyltransferase 6 6886045 NM_010283 14594 2270 1771 319 244 186 51 50 24 19 26 Ggta1: glycoprotein galactosyltransferase alpha 1, 3 6892374 NM_144786 207182 654 548 385 306 618 972 1812 922 1121 1337 Ggtl3: gamma-glutamyltransferase-like 3 6768868 NM_011820 23887 35 56 29 19 40 52 39 38 28 29 Ggtla1: gamma-glutamyltransferase-like activity 1 6791913 NM_008117 14599 11 14 38 14 27 27 18 15 15 26 Gh: growth hormone 6791443 NM_031871 80860 435 446 387 296 368 112 109 322 323 385 Ghdc: GH3 domain containing 6824007 NM_078478 66092 4361 2878 2269 2097 3647 1509 3304 1010 1755 2492 Ghitm: growth hormone inducible transmembrane protein 6833531 NM_010284 14600 492 439 231 271 473 92 69 126 48 32 Ghr: growth hormone receptor 6892512 NM_010285 14601 121 73 140 68 191 83 190 53 73 140 Ghrh: growth hormone releasing hormone 6946407 NM_001003685 14602 98 40 78 85 68 60 35 46 33 63 Ghrhr: growth hormone releasing hormone receptor 6956581 NM_021488 58991 37 30 43 22 51 28 29 9 28 37 Ghrl: ghrelin 6896360 NM_177330 208188 25 27 67 31 48 60 76 19 26 41 Ghsr: growth hormone secretagogue receptor 6868126 NM_008118 14603 14 25 28 21 15 19 26 18 19 34 Gif: gastric intrinsic factor 6946033 NM_008376 16205 75 148 26 77 43 196 114 98 107 126 Gimap1: GTPase, IMAP family member 1 6953424 NM_031247 83408 18 6 11 6 12 6 10 6 6 10 Gimap3: GTPase, IMAP family member 3 6946028 NM_174990 107526 9 56561153510Gimap4: GTPase, IMAP family member 4 6946034 NM_175035 317757 10 5 13 5 6 21 3 2 5 15 Gimap5: GTPase, IMAP family member 5 6953415 NM_153175 231931 32 15 32 21 40 242 36 23 20 42 Gimap6: GTPase, IMAP family member 6 6946031 NM_146167 231932 19 5 10 11 11 9 28 6 8 19 Gimap7: GTPase, IMAP family member 7 6946021 NM_001077410 243374 12 7 6 4 11 14 6 3 10 15 Gimap8: GTPase, IMAP family member 8 6946026 NM_174960 317758 11 31 2 5 6 14 8 6 2 16 Gimap9: GTPase, IMAP family member 9 6978386 NM_030198 78833 109 143 65 96 34 35 48 96 43 56 Gins3: GINS complex subunit 3 (Psf3 homolog) 6980991 NM_024240 109145 1213 937 509 496 614 321 394 564 968 1781 Gins4: GINS complex subunit 4 (Sld5 homolog) 6783809 NM_008119 14607 33 35 55 53 40 29 41 39 35 48 Gip: gastric inhibitory polypeptide 6977682 NM_018771 67903 1234 1156 1045 832 1007 755 1239 780 3404 3040 Gipc1: GIPC PDZ domain containing family, member 1 6910610 NM_016867 54120 128 97 10 9 16 48 31 8 7 5 Gipc2: GIPC PDZ domain containing family, member 2 6775456 NM_148951 209047 74 89 75 73 106 135 84 32 76 108 Gipc3: GIPC PDZ domain containing family, member 3 6965758 NM_001080815 381853 29 23 41 19 26 33 47 13 13 43 Gipr: gastric inhibitory polypeptide receptor 6782572 NM_001004144 216963 657 766 548 774 632 1394 915 622 1178 2683 Git1: G protein-coupled receptor kinase-interactor 1 6941217 NM_019834 26431 381 396 231 330 303 342 223 522 610 719 Git2: G protein-coupled receptor kinase-interactor 2 6971281 NM_029420 75764 405 327 446 421 689 274 239 407 367 366 Giyd2: GIY-YIG domain containing 2 6768014 NM_010288 14609 5349 3633 10198 6500 15169 653 159 56 138 289 Gja1: gap junction membrane channel protein alpha 1 6920609 NM_010289 14610 31 0 31 19 15 16 24 8 4 6 Gja10: gap junction membrane channel protein alpha 10 6788655 NM_080454 118454 33 37 23 23 22 11 26 8 680 1742 Gja12: gap junction membrane channel protein alpha 12 6925429 NM_008120 14612 235 92 97 86 316 193 212 40 33 50 Gja4: gap junction membrane channel protein alpha 4 6899791 NM_008121 14613 27 42 28 26 39 17 19 11 14 37 Gja5: gap junction membrane channel protein alpha 5 7020542 NM_001001496 414089 34 16 23 19 9 5 19 13 6 14 Gja6: gap junction membrane channel protein alpha 6 6791637 NM_008122 14615 83 123 288 382 157 412 100 5 36 38 Gja7: gap junction membrane channel protein alpha 7 6889898 NM_010290 14617 10 3265339418262853Gja9: gap junction membrane channel protein alpha 9 6894394 NM_010290 14617 34 22 17 20 39 111 102 25 26 38 Gja9: gap junction membrane channel protein alpha 9 7012866 NM_008124 14618 26 31 10 8 1 32 27 275 716 4829 Gjb1: gap junction membrane channel protein beta 1 6824932 NM_008125 14619 29 54 267 266 812 318 15 5 9 14 Gjb2: gap junction membrane channel protein beta 2 6925430 NM_008126 14620 164 42 258 50 221 14 183 5 12 43 Gjb3: gap junction membrane channel protein beta 3 6925434 NM_008127 14621 284 69 492 36 299 29 420 13 6 35 Gjb4: gap junction membrane channel protein beta 4 6925435 NM_010291 14622 53 35 73 35 45 46 47 18 21 54 Gjb5: gap junction membrane channel protein beta 5 6824940 NM_001010937 14623 41 56 312 413 6197 120 35 11 26 51 Gjb6: gap junction membrane channel protein beta 6 6942589 NM_080450 118446 11 6 14 15 26 12 18 1138 4866 6143 Gje1: gap junction membrane channel protein epsilon 1 6940219 NM_010294 14626 14 11 25 10 21 14 30 5 9 10 Gk2: glycerol kinase 2 6991561 NM_177352 235533 49 37 119 127 191 92 62 87 76 296 Gk5: glycerol kinase 5 (putative) 6813560 NM_019832 56278 347 184 241 215 287 97 65 205 253 518 Gkap1: G kinase anchoring protein 1 6955265 NM_025466 66283 101 77 125 73 118 54 59 47 31 83 Gkn1: gastrokine 1 6947692 NM_025467 66284 4 7 11 10 18 12 6944Gkn2: gastrokine 2 7019519 NM_013463 11605 569 606 874 979 529 532 606 615 554 677 Gla: galactosidase, alpha 6992632 NM_009752 12091 493 347 402 282 418 340 426 419 327 255 Glb1: galactosidase, beta 1 6944019 NM_133236 170772 405 512 510 634 517 983 678 1115 1657 1972 Glcci1: glucocorticoid induced transcript 1 6996191 NM_033320 93683 194 241 94 87 87 345 429 760 347 101 Glce: glucuronyl C5-epimerase 6872584 NM_138595 104174 1997 1431 2001 1565 3498 40 31 386 154 42 Gldc: glycine decarboxylase 6989195 NM_177350 235379 706 675 89 81 37 18 48 9 10 11 Gldn: gliomedin 6876029 NM_028923 74412 728 767 557 600 532 1058 943 664 860 1100 Gle1l: GLE1 RNA export mediator-like (yeast 6985355 NM_009149 20340 1831 948 1834 1181 1893 868 2032 1013 1098 1297 Glg1: golgi apparatus protein 1 6777932 NM_010296 14632 62 54 248 208 153 40 47 14 16 27 Gli1: GLI-Kruppel family member GLI1 6804974 NM_008130 14634 641 664 803 1010 1432 38 25 11 14 15 Gli3: GLI-Kruppel family member GLI3

Page 44 of 58 Supplemental Table S9: Selected astrocyte metabolic pathway enzymes Probe Set ID Astrocytes Astrocytes Neurons OPCs Myelin Gene Symbol Gene Title P7 P17 P16 Oligos

1428146_s_at 4374 4337 386 120 168 Acaa2 acetyl-Coenzyme A acyltransferase 2 1448987_at 6788 6623 219 853 1557 Acadl acyl-Coenzyme A dehydrogenase, long-chain 1424184_at 5125 6122 1019 2984 3471 Acadvl acyl-Coenzyme A dehydrogenase, very long chain 1422428_at 9190 16056 27 110 99 Acsbg1 acyl-CoA synthetase bubblegum family member 1 (Lipidosin) 1452771_s_at 4675 14848 1879 1516 12334 Acsl3 acyl-CoA synthetase long-chain family member 3 1451257_at 11688 21138 1837 1650 245 Acsl6 acyl-CoA synthetase long-chain family member 6 1416617_at 6017 8600 203 3015 160 Acss1 acyl-CoA synthetase short-chain family member 1 1424393_s_at 5441 6406 59 440 110 Adhfe1 alcohol dehydrogenase, iron containing, 1 1416468_at 1683 13751 167 395 634 Aldh1a1 aldehyde dehydrogenase family 1, subfamily A1 1424400_a_at 15466 13265 337 53 132 Aldh1l1 aldehyde dehydrogenase 1 family, member L1 1448143_at 3667 4335 673 1442 109 Aldh2 aldehyde dehydrogenase 2, mitochondrial 1460167_at 8887 14279 1060 2799 1781 Aldh7a1 aldehyde dehydrogenase family 7, member A1 1424714_at 5205 13855 10 135 89 Aldoc aldolase 3, C isoform 1419435_at 224 893 77 33 40 Aox1 aldehyde oxidase 1 1423844_s_at 6708 10159 175 83 93 Cbs cystathionine beta-synthase 1426243_at 3058 4372 126 28 14 Cth cystathionase (cystathionine gamma-lyase) 1419040_at 841 6309 47 49 59 Cyp2d22 cytochrome P450, family 2, subfamily d, polypeptide 22 1419559_at 1074 4043 11 42 7 Cyp4f14 cytochrome P450, family 4, subfamily f, polypeptide 14 1449316_at 1579 4853 23 21 9 Cyp4f15 cytochrome P450, family 4, subfamily f, polypeptide 15 1419456_at 1366 2265 141 308 446 Dcxr dicarbonyl L-xylulose reductase 1416049_at 4192 7765 25 922 28 Gldc glycine decarboxylase 1426235_a_at 11229 20290 466 1006 3544 Glul glutamate-ammonia ligase (glutamine synthetase) 1426522_at 7721 13454 1026 4480 3590 Hadhb hydroxyacyl-Coenzyme A dehydrogenase 1450048_a_at 5008 8734 393 2085 560 Idh2 isocitrate dehydrogenase 2 (NADP+), mitochondrial 1418238_at 3753 6262 926 1721 1041 Ivd isovaleryl coenzyme A dehydrogenase 1434499_a_at 12332 21022 8958 7440 1723 Ldhb lactate dehydrogenase B 1434354_at 1051 4896 413 66 376 Maob monoamine oxidase B 1431302_a_at 449 1314 233 95 694 Nudt7 nudix (nucleoside diphosphate linked moiety X)-type motif 7 1416383_a_at 5891 3484 538 718 1137 Pcx pyruvate carboxylase 1431012_a_at 7722 7635 638 2702 2114 Peci peroxisomal delta3, delta2-enoyl-Coenzyme A isomerase 1451149_at 2697 4882 999 1603 679 Pgm2 phosphoglucomutase 2 1417629_at 872 6932 63 82 39 Prodh proline dehydrogenase 1415673_at 4831 3873 328 1638 1968 Psph phosphoserine phosphatase 1433504_at 12268 16456 1126 770 372 Pygb brain glycogen phosphorylase 1416662_at 1039 1108 80 77 34 Sardh sarcosine dehydrogenase 1418490_at 555 1158 7 113 6 Sdsl serine dehydratase-like 1435658_at 3752 5393 223 413 195 Slc27a1 solute carrier family 27 (fatty acid transporter), member 1 1424268_at 3620 7938 495 4500 4098 Smox spermine oxidase 1435841_s_at 4332 6557 207 2577 449 Suclg2 succinate-Coenzyme A ligase, GDP-forming, beta subunit

Reference Cell Type Enriched Genes 1440142_s_at 9742 13827 31 13 77 Gfap glial fibrillary acidic protein 1448768_at 7 14 6 14 24964 Mog myelin oligodendrocyte glycoprotein 1433884_at 326 339 18197 749 239 Syt1 synaptotagmin I

Page 45 of 58 Supplemental Table S10: DNA binding proteins enriched in or expressed at high levels in astrocytes. Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Neurons OPCs Myelin Gene Symbol Gene Title Astroglia P1 P7 P17 P16 Oligos

6838183_exon 103 ---- 540 965 75 225 147 Dbx2 developing brain 2 1456258_at 573 1254 1445 1654 162 131 20 Emx2 empty spiracles homolog 2 (Drosophila) 1459211_at 1239 1717 1631 1632 55 10 15 Gli2 GLI-Kruppel family member GLI2 1456067_at 1254 3124 1787 3417 92 23 17 Gli3 GLI-Kruppel family member GLI3 1418102_at 894 1321 4237 3253 477 486 17 Hes1 hairy and enhancer of split 1 (Drosophila) 1456010_x_at 37 13661 15031 10385 60 6885 77 Hes5 hairy and enhancer of split 5 (Drosophila) 1415999_at 4335 3943 4758 5686 1247 2965 457 Hey1 hairy/enhancer-of-split related with YRPW motif 1 1428662_a_at 4576 18891 14949 31097 1456 507 756 Hod homeobox only domain 1425895_a_at 1703 526 2216 834 139 67 49 Id1 inhibitor of DNA binding 1 1435176_a_at 7756 21198 17391 13261 8463 3040 458 Id2 inhibitor of DNA binding 2 1416630_at 11160 5694 13298 9320 125 573 178 Id3 inhibitor of DNA binding 3 1423259_at 13209 13597 14060 24862 882 551 174 Id4 inhibitor of DNA binding 4 1448181_at 2020 2216 3564 5024 261 706 187 Klf15 Kruppel-like factor 15 1418317_at 1226 1978 3398 3501 309 413 23 Lhx2 LIM homeobox protein 2 1424596_s_at 256 1050 1684 2170 29 3702 10 Lmcd1 LIM and cysteine-rich domains 1 1440167_s_at 4523 4939 2004 4471 107 1144 119 Lpp LIM domain containing preferred translocation partner in lipoma 1416543_at 8970 1102 1720 2773 90 126 1590 Nfe2l2 nuclear factor, erythroid derived 2, like 2 1429138_at 1761 6161 5315 3935 384 2591 164 Npas3 neuronal PAS domain protein 3 1434921_at 343 479 1062 1128 5 450 8 Nr2e1 subfamily 2, group E, member 1 1416149_at 16211 24081 20693 13158 210 30004 33106 Olig1 oligodendrocyte 1 1416232_at 641 1628 1797 491 28 8670 3283 Olig2 oligodendrocyte transcription factor 2 1437601_at 480 416 819 599 115 99 7 Otx1 orthodenticle homolog 1 (Drosophila) 1419271_at 939 1928 1704 1281 74 98 8 Pax6 paired box gene 6 1451132_at 11084 3922 8444 13306 317 337 374 Pbxip1 pre-B-cell leukemia transcription factor interacting protein 1 1435197_at 6434 12352 11610 12073 1524 9267 5005 Pou3f3 POU domain, class 3, transcription factor 3 1440870_at 1532 2436 1852 1695 127 23 28 Prdm16 PR domain containing 16 1425528_at 391 1149 652 1098 24 1275 46 Prrx1 paired related homeobox 1 1417877_at 147 1641 1896 376 283 75 62 Riken RIKEN cDNA 2310005P05 gene 1436931_at 1116 4121 3880 4535 35 1047 16 Rfx4 regulatory factor X, 4 (influences HLA class II expression) 1452386_at 104 984 650 667 8 695 12 Sall3 sal-like 3 (Drosophila) 6974082_exon 288 ---- 537 649 122 64 61 Sox1 SRY-box containing gene 1 1416967_at 6826 14401 11544 7754 762 5235 1674 SRY-box containing gene 2 1437059_at 1645 2832 4645 4499 178 3130 292 Sox21 SRY-box containing gene 21 1424950_at 1597 2893 1854 1426 103 264 15 Sox9 SRY-box containing gene 9 1426690_a_at 3039 6632 7878 10228 354 1415 1270 Srebf1 sterol regulatory element binding factor 1 1418091_at 3435 56 72 921 50 27 22 Tcfcp2l1 transcription factor CP2-like 1 1434400_at 256 1292 1419 267 98 222 184 Tgif2 TGFB-induced factor 2 1450644_at 4871 19009 9210 10164 267 10403 145 Zfp36l1 protein 36, C3H type-like 1 6822317_exon 330 ---- 489 548 74 36 15 Zic2 Zic finger protein of the cerebellum 2

Reference Cell Type Enriched Genes 1440142_s_at 16201 15463 9742 13827 31 13 77 Gfap glial fibrillary acidic protein 1448768_at 12 2 7 14 6 14 24964 Mog myelin oligodendrocyte glycoprotein 1433884_at 315 306 326 339 18197 749 239 Syt1 synaptotagmin I

Page 46 of 58 Supplemental Table S11: Selected astrocyte expressed receptors. Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Neurons OPCs Myelin Gene Symbol Gene Title Astroglia P1 P7 P17 P16 Oligos

1429022_at 301 11776 14803 16574 1756 9520 249 Adcyap1r1 adenylate cyclase activating polypeptide 1 receptor 1 1423586_at 1912 958 2145 2598 52 341 43 Axl AXL receptor tyrosine kinase 1425492_at 7784 14510 10477 10204 954 2603 1077 Bmpr1a bone morphogenetic protein receptor, type 1A 1437312_at 965 4364 4848 6628 244 54 23 Bmpr1b bone morphogenetic protein receptor, type 1B 1434310_at 8338 14751 9363 10502 7983 7260 6263 Bmpr2 bone morphogenic protein receptor, type II 1456226_x_at 16632 23791 11805 3492 2170 3672 23621 Ddr1 discoidin domain receptor family, member 1 1423571_at 642 1459 3743 7906 124 399 121 Edg1 endothelial differentiation sphingolipid G-protein-coupled receptor 1 1437347_at 3374 20052 20308 19381 386 15116 300 Ednrb endothelin receptor type B 1435888_at 479 4714 4909 3454 309 72 34 Egfr epidermal growth factor receptor (Erbb1) 6784005_exon 534 ---- 759 436 26 21 31 Erbb2 Erbb2: v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 1428011_a_at 5644 7694 3992 2550 647 1561 6940 Erbb2ip Erbb2 interacting protein 1421841_at 2925 9511 12169 18684 376 569 196 Fgfr3 fibroblast growth factor receptor 3 1437284_at 3411 1161 2400 4119 449 818 98 Fzd1 frizzled homolog 1 (Drosophila) 1418532_at 2587 1532 2956 7546 50 232 31 Fzd2 frizzled homolog 2 (Drosophila) 1455021_at 1036 7830 13482 15805 10179 4625 3250 Gabbr1 gamma-aminobutyric acid (GABA-B) receptor, 1 1439015_at 975 537 1597 3551 773 5882 2622 Gfra1 glial cell line derived neurotrophic factor family receptor alpha 1 1454685_at 2069 474 1270 3968 117 420 661 Gpr146 G protein-coupled receptor 146 1424146_at 4872 2729 18491 31275 293 17708 7780 Gpr37l1 G protein-coupled receptor 37-like 1 1433485_x_at 748 2500 7698 5011 382 1871 517 Gpr56 G protein-coupled receptor 56 1449245_at 35 27 997 8611 106 42 18 Grin2c glutamate receptor, ionotropic, NMDA2C (epsilon 3) 1417423_at 8747 1485 5587 13357 8069 6609 2239 Grina glutamate receptor, ionotropic, NMDA-associated protein 1 1430136_at 194 12186 11778 16185 1594 140 277 Grm3 glutamate receptor, metabotropic 3 1430136_at 194 12186 11778 16185 1594 140 277 Grm3 glutamate receptor, metabotropic 3 1417505_s_at 1411 1165 1905 2520 863 199 49 Il11ra1 interleukin 11 receptor, alpha chain 1 1452843_at 4877 2164 4334 4329 623 2343 729 Il6st interleukin 6 signal transducer (gp130) 1418393_a_at 665 962 1569 1454 169 46 42 Itga7 integrin alpha 7 1452784_at 9344 15219 9018 10595 1757 10037 4162 Itgav integrin alpha V 1417533_a_at 5197 7125 8569 6783 193 1036 231 Itgb5 integrin beta 5 1433891_at 1383 1191 1686 4206 637 271 211 Lgr4 leucine-rich repeat-containing G protein-coupled receptor 4 1427028_at 121 326 460 443 120 71 91 Lgr6 leucine-rich repeat-containing G protein-coupled receptor 6 1422869_at 620 1457 1948 7370 59 569 89 Mertk c-mer proto-oncogene tyrosine kinase 1418634_at 3933 10032 5909 6832 147 5033 869 Notch1 Notch gene homolog 1 (Drosophila) 1455556_at 2351 3459 4570 3060 234 170 352 Notch2 Notch gene homolog 2 (Drosophila) 1421965_s_at 396 3380 2206 1480 118 66 106 Notch3 Notch gene homolog 3 (Drosophila) 1420838_at 15046 9307 16457 29189 903 10544 1796 Ntrk2 neurotrophic tyrosine kinase, receptor, type 2 1433825_at 170 2896 2418 1497 985 3602 38 Ntrk3 Neurotrophic tyrosine kinase, receptor, type 3 1417151_a_at 610 1268 11407 36602 382 702 362 Ntsr2 neurotensin receptor 2 1436970_a_at 1739 1147 1932 2566 76 22 10 Pdgfrb platelet derived growth factor receptor, beta polypeptide 1435254_at 2088 4719 6380 8752 185 1828 988 Plxnb1 plexin B1 1428853_at 3898 12783 12322 12193 929 3310 3066 Ptch1 patched homolog 1 1418690_at 311 32945 23256 13598 115 16531 126 Ptprz1 protein tyrosine phosphatase, receptor type Z, polypeptide 1 1451789_a_at 1203 926 1543 1450 94 964 658 Ryk receptor-like tyrosine kinase 1427049_s_at 2055 2172 3523 2714 94 596 57 Smo smoothened homolog (Drosophila) 1422782_s_at 1878 720 1960 5538 36 122 117 Tlr3 toll-like receptor 3 1448147_at 954 9924 13530 13298 638 3063 115 Tnfrsf19 tumor necrosis factor receptor superfamily, member 19 (Troy)

Reference Cell Type Enriched Genes 1440142_s_at 16201 15463 9742 13827 31 13 77 Gfap glial fibrillary acidic protein 1448768_at 12 2 7 14 6 14 24964 Mog myelin oligodendrocyte glycoprotein 1433884_at 315 306 326 339 18197 749 239 Syt1 synaptotagmin I

Page 47 of 58 Supplemental Table S12: Genes enriched in astrocytes Probe Set ID Cultured Astrocytes Astrocytes Astrocytes Neurons OPCs Myelin Gene Symbol Gene Title Astroglia P1 P7 P17 P16 Oligos

1434449_at 13089 22366 22900 24431 195 192 345 Aqp4 aquaporin 4 1436878_at 756 325 2755 4561 703 626 91 Baalc brain and acute leukemia, cytoplasmic 1433653_at 8 195 1358 3710 10 3 6 BC029169 Fam20a (cDNA sequence BC029169) 1434203_at 14291 362 2063 29104 392 329 571 BC055107 Drr1 (down regulated in renal cell carcinoma 1), Tu3a, Fam107a 1424186_at 12423 17684 13977 8637 404 185 64 Ccdc80 coiled-coil domain containing 80 1426147_s_at 273 642 4856 7485 70 357 53 Cldn10 claudin 10 1418457_at 523 510 2572 4069 383 1003 9 Cxcl14 chemokine (C-X-C motif) ligand 14 1449316_at 311 1150 1579 4853 23 21 9 Cyp4f15 cytochrome P450, family 4, subfamily f, polypeptide 15 1438133_a_at 5067 5462 9885 6461 515 688 17 Cyr61 cysteine rich protein 61 (Ccn1) 1448669_at 3076 5268 6198 9127 3121 180 33 Dkk3 dickkopf homolog 3 (Xenopus laevis) 1417408_at 5303 2485 5596 10828 122 881 44 F3 coagulation factor III 1423136_at 2466 1106 2259 6905 325 186 1041 Fgf1 fibroblast growth factor 1 1426509_s_at 5958 413 3988 3541 3 25 31 Gfap glial fibrillary acidic protein 1440142_s_at 16201 15463 9742 13827 31 13 77 Gfap glial fibrillary acidic protein 1448397_at 276 125 1639 17089 159 73 112 Gjb6 gap junction membrane channel protein beta 6 1438020_at 36 442 6134 9242 1277 493 12 Hapln1 hyaluronan and proteoglycan link protein 1 1418350_at 4719 1430 2377 3098 295 874 250 Hbegf heparin-binding EGF-like growth factor 1429023_at 1148 565 1505 2765 37 682 1296 Hepacam hepatocyte cell adhesion molecule 1417043_at 2749 723 4162 8749 60 147 124 Lcat lecithin cholesterol acyltransferase 1434210_s_at 2273 10236 8518 5419 224 243 215 Lrig1 leucine-rich repeats and immunoglobulin-like domains 1 1416006_at 583 2582 6200 4158 156 1935 104 Mdk midkine 1448139_at 17672 18515 25685 36255 71 70 202 Mlc1 megalencephalic leukoencephalopathy with subcortical cysts 1 1423086_at 666 2316 2293 2626 172 2970 3372 Npc1 Niemann Pick type C1 1449351_s_at 2348 1381 1968 2004 337 926 2968 Pdgfc platelet-derived growth factor, C polypeptide 1430700_a_at 4758 8192 20076 23333 74 1113 103 Pla2g7 phospholipase A2, group VII 1437030_at 3583 738 2298 4646 20 35 11 Plcd4 phospholipase C, delta 4 1416211_a_at 156 11973 14181 10587 618 5082 7196 Ptn pleiotrophin 1457800_at 459 3746 9123 20768 451 287 127 Riken RIKEN cDNA 2900019G14 gene 1436231_at 307 1676 1596 7636 8 16 64 Riken RIKEN cDNA 2900052N01 gene 1434342_at 345 330 2136 9066 204 3594 10802 S100b S100 protein, beta polypeptide, neural 1451627_a_at 436 552 3500 9010 224 127 29 Slc1a2 solute carrier family 1, member 2, Glt-1, EAAT2 1452031_at 4380 25844 24681 21057 216 2274 240 Slc1a3 solute carrier family 1, member 3, GLAST, EAAT1 1452132_at 1263 2929 6608 7011 102 1988 134 Tlcd1 TLC domain containing 1 1420514_at 10205 10847 10538 12771 2514 617 162 Tmem47 Transmembrane protein 47 1420834_at 804 477 1025 1170 10299 1430 1299 Vamp2 vesicle-associated membrane protein 2 1437708_x_at 13629 16647 10115 12950 1203 14167 33447 Vamp3 vesicle-associated membrane protein 3 1434796_at 2039 2149 3372 3798 3598 3028 1448 Vamp4 vesicle-associated membrane protein 4 1448162_at 3960 15877 15769 16127 229 3068 96 Vcam1 vascular cell adhesion molecule 1

Reference Cell Type Enriched Genes 1440142_s_at 16201 15463 9742 13827 31 13 77 Gfap glial fibrillary acidic protein 1448768_at 12 2 7 14 6 14 24964 Mog myelin oligodendrocyte glycoprotein 1433884_at 315 306 326 339 18197 749 239 Syt1 synaptotagmin I

Page 48 of 58 Supplemental Table S13: Astrocyte expressed signals that might control endothelial development and motility Probe Set ID Astrocytes Astrocytes Neurons OPCs Myelin Gene Symbol Gene Title P7 P17 P16 Oligos

1423396_at 4017 7131 25 64 78 Agt angiotensinogen (serpin peptidase inhibitor, clade A, member 8) 1454890_at 6175 5491 356 417 142 Amot angiomotin 1428785_at 966 1394 446 644 182 Amotl1 angiomotin-like 1 1439066_at 1960 734 220 54 85 Angpt1 angiopoietin 1 1451038_at 318 1781 310 141 582 Apln apelin 1424677_at 2649 6081 49 2098 331 Cyp2j9 cytochrome P450, family 2, subfamily j, polypeptide 9 1416039_x_at 21784 16096 1064 1477 77 Cyr61 cysteine rich protein 61 6928939_exon 959 918 44 10 23 Hgf hepatocyte growth factor 1420911_a_at 25011 31089 617 3283 754 Mfge8 milk fat globule-EGF factor 8 protein 1449251_at 1202 1848 146 423 128 Ndph Norrie disease homolog 1456532_at 524 917 22 30 19 Pdgfd Platelet-derived growth factor, D polypeptide 1416589_at 7035 11387 417 2574 1038 Sparc secreted acidic cysteine rich glycoprotein 1420909_at 2043 2144 366 871 311 Vegfa vascular endothelial growth factor A 1451803_a_at 1201 1110 379 844 81 Vegfb vascular endothelial growth factor B 1439766_x_at 1247 855 199 178 12 Vegfc vascular endothelial growth factor C

Reference Cell Type Enriched Genes 1440142_s_at 9742 13827 31 13 77 Gfap glial fibrillary acidic protein 1448768_at 7 14 6 14 24964 Mog myelin oligodendrocyte glycoprotein 1433884_at 326 339 18197 749 239 Syt1 synaptotagmin I

Page 49 of 58 Supplemental Table S14: Oligodendrocyte synthesis of creatine and carnitine Probe Set ID Astrocytes Neurons OPCs Oligos Myelin Gene Symbol Gene Title P17 P16 Oligos

1415776_at 1104 595 1112 1153 1242 Aldh3a2 (1.2.1.3) aldehyde dehydrogenase family 3, subfamily A2 1437398_a_at 626 201 724 860 1264 Aldh9a1 (1.2.1.47) aldehyde dehydrogenase 9, subfamily A1 1457268_at 640 442 901 740 671 Dot1l (2.1.1.43) DOT1-like, histone H3 methyltransferase (S. cerevisiae) 1422558_at 479 214 143 3476 7119 Gamt (2.1.1.2) guanidinoacetate methyltransferase 1423569_at 1601 343 4441 6036 10115 Gatm (2.1.4.1) glycine amidinotransferase (L-arginine:glycine amidinotransferase) 1420725_at 194 187 253 262 494 Tmlhe (1.14.11.8) trimethyllysine hydroxylase, epsilon

Reference Cell Type Enriched Genes 1440142_s_at 13827 31 13 47 77 Gfap glial fibrillary acidic protein 1448768_at 14 6 14 5983 24964 Mog myelin oligodendrocyte glycoprotein 1433884_at 339 18197 749 416 239 Syt1 synaptotagmin I

Page 50 of 58 Supplemental Table S15 First Page: Genes statistically enriched in developing astrocytes. Probe Set ID Gene Name Fold Enriched In Developing Astrocytes Over Mature Astrocytes 1 1454694_a_at Top2a 20.2 2 1451246_s_at Aurkb 13.9 3 1448314_at Cdc2a 12.9 4 1448627_s_at Pbk 12.6 5 1416529_at Emp1 12.2 6 1427682_a_at Egr2 11.1 7 1426817_at Mki67 10.9 8 1434819_at St6gal2 10.8 9 1439904_at Fstl5 10.1 10 1416558_at Melk 9.5 11 1436847_s_at Cdca8 9.5 12 1427122_at Copg2as2 8.8 13 1420994_at B3gnt5 8.7 14 1433935_at AU020206 8.5 15 1429295_s_at Trip13 7.9 16 1448205_at Ccnb1-rs1 7.5 17 1417910_at Ccna2 7.5 18 1452954_at Ube2c 7.4 19 1453006_at Fgfbp3 7.1 20 1455980_a_at Gas2l3 7.0 21 1415810_at Uhrf1 6.9 22 1448182_a_at Cd24a 6.7 23 1438852_x_at Mcm6 6.5 24 1419513_a_at Ect2 6.5 25 1416266_at Pdyn 6.5 26 1456509_at 1110032F04Rik 6.5 27 1435415_x_at Marcksl1 6.5 28 1424278_a_at Birc5 6.5 29 1419153_at 2810417H13Rik 6.4 30 1424046_at Bub1 6.3 31 1438009_at Hist1h2ao 6.3 32 1447363_s_at Bub1b 6.1 33 1427161_at Cenpf 6.0 34 1417822_at D17H6S56E-5 6.0 35 1450920_at Ccnb2 6.0 36 1436808_x_at Mcm5 6.0 37 1416410_at Pafah1b3 5.7 38 1455034_at Nr4a2 5.7 39 1436013_at Gsg1l 5.6 40 1435306_a_at Kif11 5.5 41 1452242_at Cep55 5.5 42 1417457_at Cks2 5.5 43 1457140_s_at 4632411J06Rik 5.4 44 1436329_at Egr3 5.3 45 1460627_at Thsd7b 5.3 46 1448777_at Mcm2 5.2 47 1418666_at Ptx3 5.2 48 1450842_a_at Cenpa 5.2 49 1434400_at Tgif2 5.2 50 1447914_x_at 4833411O04Rik 5.2 51 1454613_at 9430041P20Rik 5.2 52 1423775_s_at Prc1 5.2 53 1442280_at D2Ertd750e 5.2 54 1416846_a_at Pdzrn3 5.2 55 1417877_at 2310005P05Rik 5.2 56 1417262_at Ptgs2 5.1 57 1448194_a_at H19 5.1 58 1423499_at Sncaip 5.1 59 1416342_at Tnc 5.1 60 1415972_at Marcks 5.0 61 1455272_at Grm5 5.0 62 1416299_at Shcbp1 5.0 63 1451128_s_at Kif22 5.0 64 1436905_x_at Laptm5 5.0 65 1424118_a_at Spbc25 5.0 66 1435963_at Sema5b 5.0 67 1416405_at Bgn 4.8 68 1434767_at C79407 4.8 69 1428105_at Tpx2 4.8 70 1449171_at Ttk 4.7 71 1416118_at Trim59 4.7 72 1418507_s_at Socs2 4.7 73 1421546_a_at Racgap1 4.7 74 1457446_at Opcml 4.7 75 1457270_at Rcvrn 4.7 76 1436907_at Nav1 4.6 77 1418334_at Dbf4 4.6 78 1436392_s_at Tcfap2c 4.6 79 1449401_at C1qc 4.6 80 1416698_a_at Cks1b 4.5

Page 51 of 58 Supplemental Table S16 First Page: Genes statistically enriched in mature astrocytes. Probe Set ID Gene Name Fold Enriched In Mature Astrocytes Over Developing Astrocytes 1 1452975_at Agxt2l1 34.6 2 1427345_a_at Sult1a1 25.0 3 1443745_s_at Dmp1 16.0 4 1434202_a_at BC055107 14.4 5 1421425_a_at Dscr1l1 13.0 6 1429166_s_at Clmn 12.6 7 1418091_at Tcfcp2l1 11.2 8 1424208_at Ptger4 10.2 9 1448397_at Gjb6 9.7 10 1435314_at Tph2 9.5 11 1454969_at Lypd6 8.8 12 1424553_at Hhatl 8.1 13 1451174_at Lrrc33 8.0 14 1419665_a_at Nupr1 7.3 15 1423183_at Lgi1 7.2 16 1436698_x_at BC054438 7.2 17 1435741_at Pde8b 6.9 18 1434601_at Amigo2 6.9 19 1416468_at Aldh1a1 6.6 20 1448602_at Pygm 6.3 21 1452385_at Usp53 6.1 22 1417629_at Prodh 6.0 23 1449245_at Grin2c 5.9 24 1455304_at Unc13c 5.8 25 1428536_at Kcng4 5.8 26 1439830_at Map3k5 5.7 27 1419874_x_at AI467657 5.5 28 1441867_x_at 4930534B04Rik 5.5 29 1457495_at 2900052N01Rik 5.5 30 1419040_at Cyp2d22 5.5 31 1424854_at Hist1h4i 5.4 32 1437418_at Dmt2 5.4 33 1456633_at Trpm3 5.3 34 1427126_at Hspa1b 5.1 35 1446708_at Hif3a 5.0 36 1437759_at Pfkp 5.0 37 1435957_at B830032F12 4.9 38 1422155_at Hist2h3c2 4.9 39 1437056_x_at Crispld2 4.9 40 1423306_at 2010002N04Rik 4.9 41 1449027_at Rhou 4.8 42 1428386_at Acsl3 4.8 43 1419435_at Aox1 4.7 44 1418212_at Omg 4.7 45 1454678_s_at A130022J15Rik 4.6 46 1429900_at 5330406M23Rik 4.6 47 1438756_at Ankrd29 4.6 48 1439163_at Zbtb16 4.5 49 1421037_at Npas2 4.4 50 1422869_at Mertk 4.4 51 1424842_a_at Arhgap24 4.3 52 1451038_at Apln 4.2 53 1436011_at Elmo2 4.2 54 1434474_at Abca5 4.2 55 1451361_a_at Pnpla7 4.1 56 1436569_at C030045D06Rik 4.0 57 1455799_at Rorb 4.0 58 1416619_at 4632428N05Rik 4.0 59 1428394_at Phyhd1 4.0 60 1428923_at Ppp1r3g 3.9 61 1448690_at Kcnk1 3.9 62 1417702_a_at Hnmt 3.9 63 1439527_at Pgr 3.8 64 1428223_at Mfsd2 3.8 65 1423414_at Ptgs1 3.8 66 1427183_at Efemp1 3.8 67 1417373_a_at Tuba4a 3.8 68 1434354_at Maob 3.8 69 1436216_s_at 2610204M08Rik 3.8 70 1438967_x_at Amhr2 3.7 71 1452280_at Farp1 3.7 72 1433888_at Atp2b2 3.6 73 1417765_a_at Amy1 3.6 74 1455460_at 6430547I21Rik 3.6 75 1458379_at 9330159F19Rik 3.6 76 1416455_a_at Cryab 3.6 77 1449641_at Adk 3.5 78 1419383_at S100b 3.5 79 1422477_at Cables1 3.5 80 1460061_at Adra1a 3.5

Page 52 of 58 Supplemental Table S17 First Page: Genes statistically enriched in OPCs. Probe Set ID Gene Name Fold Enriched In OPCs Over Myelin OLs 1 1421917_at Pdgfra 137.1 2 1418310_a_at Rlbp1 96.1 3 1440884_s_at A530047J11Rik 83.3 4 1450251_a_at Lnx1 83.1 5 1423341_at Cspg4 81.8 6 1454613_at 9430041P20Rik 79.2 7 1436948_a_at 6430550H21Rik 75.9 8 1448842_at Cdo1 74.3 9 1436718_at Nxph1 69.8 10 1435790_at Olfm2 67.9 11 1456354_at Chrna4 66.9 12 1425745_a_at Tacc2 59.0 13 1436733_at E130309F12Rik 58.3 14 1438306_at Rnf180 56.5 15 1460627_at Thsd7b 56.4 16 1418690_at Ptprz1 56.2 17 1450713_at Cspg5 55.6 18 1418464_at Matn4 55.0 19 1428089_at Slitrk1 50.7 20 1451342_at Spon1 50.5 21 1424850_at Map3k1 49.9 22 1435190_at AI465420 49.9 23 1449533_at Tmem100 49.3 24 1424701_at Pcdh20 49.3 25 1437308_s_at F2r 48.2 26 1428902_at Chst11 47.4 27 1448627_s_at Pbk 46.5 28 1452065_at Vstm2 45.0 29 1426204_a_at Oprl1 43.9 30 1449581_at Emid1 42.8 31 1434759_at Lrrtm3 42.4 32 1428142_at Etv5 42.2 33 1449154_at Col11a1 42.1 34 1436719_at Slc35f1 40.9 35 1417419_at Ccnd1 40.8 36 1415877_at Dpysl3 40.0 37 1434430_s_at Adora2b 39.5 38 1439904_at Fstl5 39.3 39 1433716_x_at Gfra2 39.0 40 1434437_x_at Rrm2 38.5 41 1434685_at D3Bwg0562e 38.3 42 1450975_at Cacng4 37.9 43 1435672_at 3830612M24 37.6 44 1417022_at Slc7a3 36.7 45 1456010_x_at Hes5 36.3 46 1438511_a_at 1190002H23Rik 36.3 47 1456283_at Neto1 35.8 48 1436999_at 5033414K04Rik 35.5 49 1435720_at Kcnd3 34.8 50 1426817_at Mki67 34.7 51 1422835_at Kcnd2 34.1 52 1427482_a_at Car8 33.3 53 1455400_at Ddah1 33.1 54 1434921_at Nr2e1 33.0 55 1422649_at Cntn6 32.7 56 1425814_a_at Calcrl 32.4 57 1447551_x_at Lphn3 32.2 58 1453125_at Sox11 32.2 59 1422552_at Rprm 32.2 60 1434993_at Zbed4 32.0 61 1454694_a_at Top2a 31.9 62 1448655_at Lrp1 31.7 63 1435162_at Prkg2 31.1 64 1434891_at Ptgfrn 30.6 65 1426858_at Inhbb 30.0 66 1437528_x_at A730017C20Rik 29.6 67 1455521_at Klf12 29.6 68 1454869_at Wdr40b 29.5 69 1438007_at AI851790 29.5 70 1435765_at E130114P18Rik 29.4 71 1434728_at Gria3 29.3 72 1456512_at Pdzrn4 29.2 73 1450644_at Zfp36l1 29.0 74 1453282_at Cxadr 28.9 75 1438200_at Sulf1 28.5 76 1420965_a_at Enc1 28.4 77 1439774_at Prrx1 27.8 78 1448162_at Vcam1 27.6 79 1450027_at Sdc3 27.3 80 1454974_at Ntn1 27.2

Page 53 of 58 Supplemental Table S18: Genes statistically enriched myelinating OLs compared to OPCs. Probe Set ID Gene Name Fold Enriched In Myelin OLs Over OPCs 1 1417275_at Mal 421.1 2 1448768_at Mog 312.6 3 1418086_at Ppp1r14a 241.2 4 1456174_x_at Ndrg1 200.8 5 1433785_at Mobp 192.9 6 1442075_at AI314604 188.4 7 1440902_at A330104H05Rik 144.8 8 1441909_s_at 9530066K23Rik 119.2 9 1416003_at Cldn11 117.5 10 1435854_at Tmem10 102.5 11 1419529_at Il23a 95.1 12 1455123_at St18 93.9 13 1417143_at Edg2 90.8 14 1438785_at Enpp6 87.6 15 1418472_at Aspa 87.6 16 1460219_at Mag 84.2 17 1434094_at 6330530A05Rik 81.3 18 1450875_at Gpr37 81.1 19 1423946_at Pdlim2 75.8 20 1418739_at Sgk2 73.0 21 1447914_x_at 4833411O04Rik 71.5 22 1448416_at Mgp 70.7 23 1416371_at Apod 68.3 24 1420760_s_at Ndrl 67.9 25 1449365_at Edg8 66.7 26 1429901_at Tcba1 65.4 27 1455965_at Adamts4 63.2 28 1448507_at Efhd1 61.9 29 1449383_at Adssl1 61.7 30 1447807_s_at Plekhh1 58.4 31 1442138_at Gpr62 57.0 32 1425468_at Plp1 54.2 33 1450483_at Gja12 52.4 34 1456228_x_at Mbp 50.5 35 1453269_at Unc5b 50.2 36 1448752_at Car2 48.8 37 1420962_at Hapln2 47.7 38 1437846_x_at Bace2 47.2 39 1416713_at 2700055K07Rik 47.1 40 1449123_at Itih3 44.3 41 1434500_at Ttyh2 44.0 42 1430030_at 5330426P16Rik 43.4 43 1417574_at Cxcl12 43.0 44 1428599_at Kndc1 42.4 45 1424987_at 5430435G22Rik 40.5 46 1452834_at 2600010E01Rik 39.9 47 1454733_at Nod1 39.7 48 1425546_a_at Trf 38.1 49 1433667_at Lgi3 37.7 50 1435849_at 6330417G02Rik 36.8 51 1439785_at 9630013A20Rik 36.4 52 1435463_s_at Myo1d 35.5 53 1438672_at Parvb 35.5 54 1434399_at Galnt6 35.4 55 1427420_at Nkx6-2 35.4 56 1453009_at Cpm 35.1 57 1450725_s_at Car14 35.0 58 1448729_a_at Sept4 34.9 59 1422558_at Gamt 33.6 60 1451431_a_at Dbndd2 33.0 61 1426663_s_at Slc45a3 31.9 62 1441969_at Trim36 31.5 63 1439078_at Klhl4 30.7 64 1450813_a_at Tnni1 30.6 65 1436974_at A230069A22Rik 30.6 66 1435125_at BB120497 30.5 67 1457323_at C030030A07Rik 30.1 68 1457270_at Rcvrn 29.6 69 1440354_at Elovl7 29.4 70 1423871_at Tmem63a 28.5 71 1424567_at Tspan2 28.4 72 1417110_at Man1a 28.4 73 1449151_at Pctk3 27.7 74 1428899_at Tmem182 26.6 75 1434432_at Rffl 26.5 76 1426215_at Ddc 24.9 77 1424378_at Ldlrap1 24.8 78 1438428_at Jph1 24.6 79 1428750_at Cdc42ep2 24.0 80 1417222_a_at Tmem123 23.2

Page 54 of 58 Supplemental Table S19 First Page: Genes statistically enriched in MOG+ myelinating OLs compared to MOG- OLs. Probe Set ID Gene Name Fold Enriched In MOG positive Myelin OLs Over MOG Negative OLs 1 1449979_a_at Spock3 9.8 2 1420962_at Hapln2 9.6 3 1456174_x_at Ndrg1 9.2 4 1430118_at 2700046A07Rik 8.9 5 1447914_x_at 4833411O04Rik 8.7 6 1439648_at Anln 8.7 7 1428696_at Rftn1 8.5 8 1416424_at M6prbp1 8.4 9 1434628_a_at Rhpn2 8.3 10 1416371_at Apod 7.8 11 1427996_at BC028528 7.6 12 1448507_at Efhd1 7.4 13 1452834_at 2600010E01Rik 7.4 14 1428509_at Myo1e 7.3 15 1430291_at Dock5 7.2 16 1449383_at Adssl1 7.1 17 1448767_s_at Gjb1 7.1 18 1434641_x_at Sez6l2 7.0 19 1448942_at Gng11 7.0 20 1435264_at Emilin2 7.0 21 1439078_at Klhl4 6.6 22 1452250_a_at Col6a2 6.6 23 1418472_at Aspa 6.6 24 1448982_at Klk6 6.5 25 1420760_s_at Ndrl 6.4 26 1435165_at Cntn2 6.4 27 1445597_s_at Hrasls3 6.2 28 1418086_at Ppp1r14a 6.1 29 1436216_s_at 2610204M08Rik 6.0 30 1428397_at B3galt5 6.0 31 1416318_at Serpinb1a 5.9 32 1451038_at Apln 5.9 33 1452067_at Asahl 5.9 34 1455056_at Lmo7 5.9 35 1418402_at Adam19 5.8 36 1450686_at Pon2 5.8 37 1451348_at Depdc6 5.6 38 1427146_at AI790298 5.4 39 1450154_at Folh1 5.3 40 1452277_at Arsg 5.3 41 1418424_at Tnfaip6 5.3 42 1451411_at Gprc5b 5.1 43 1438628_x_at Cntn3 5.1 44 1434136_at 6332401O19Rik 5.0 45 1416304_at Litaf 5.0 46 1429830_a_at Cd59a 5.0 47 1417273_at Pdk4 5.0 48 1422754_at Tmod1 4.9 49 1448918_at Slco3a1 4.8 50 1425217_a_at Synj2 4.8 51 1457323_at C030030A07Rik 4.7 52 1433801_at 9930012K11Rik 4.7 53 1455915_at Galnt4 4.6 54 1422629_s_at Shroom3 4.6 55 1417275_at Mal 4.6 56 1448416_at Mgp 4.6 57 1426663_s_at Slc45a3 4.6 58 1435959_at Arhgap15 4.6 59 1437404_at Mast4 4.5 60 1433596_at Dnajc6 4.5 61 1442652_at Gm410 4.5 62 1448113_at Stmn1 4.5 63 1418912_at Plxdc2 4.5 64 1434191_at A530016O06Rik 4.5 65 1426541_a_at Endod1 4.4 66 1456684_at Tmem74 4.4 67 1429055_at 4930506M07Rik 4.3 68 1426285_at Lama2 4.3 69 1416713_at 2700055K07Rik 4.3 70 1452141_a_at Sepp1 4.3 71 1447807_s_at Plekhh1 4.3 72 1428615_at P2ry5 4.2 73 1428750_at Cdc42ep2 4.2 74 1427387_a_at Itgb4 4.2 75 1455663_at Olfml1 4.2 76 1448539_a_at Acy3 4.2 77 1450241_a_at Evi2a 4.2 78 1455435_s_at Chdh 4.2 79 1426258_at Sorl1 4.2 80 1445565_at Hist1h2be 4.1

Page 55 of 58 Supplemental Table S20 First Page: Genes statistically enriched in in vivo astrocytes compared to cultured astroglia. Probe Set ID Gene Name Fold Enriched In In Vivo Astrocytes Over Cultured Astroglia 1 1457536_at Gpc5 97.0 2 1438020_at Hapln1 83.5 3 1456010_x_at Hes5 70.4 4 1438751_at Slc30a10 61.4 5 1460408_at Gabrg1 60.5 6 1449038_at Hsd11b1 53.3 7 1436115_at Gm266 52.8 8 1421093_at Slc7a10 52.5 9 1418937_at Dio2 52.0 10 1419420_at St6galnac5 48.9 11 1429651_at Phactr3 47.1 12 1455444_at Gabra2 43.2 13 1428923_at Ppp1r3g 41.5 14 1455460_at 6430547I21Rik 41.3 15 1449147_at Chst1 40.7 16 1448397_at Gjb6 38.0 17 1449245_at Grin2c 35.9 18 1428303_at 1500005I02Rik 35.5 19 1435583_at 9330182L06Rik 33.1 20 1417051_at Pcdh8 30.8 21 1418212_at Omg 30.3 22 1433653_at BC029169 29.6 23 1440973_at LOC552874 28.5 24 1455304_at Unc13c 27.5 25 1428922_at 1200009O22Rik 27.2 26 1416444_at Elovl2 27.1 27 1430136_at Grm3 26.3 28 1442700_at Pde4b 26.0 29 1424701_at Pcdh20 25.0 30 1419332_at Egfl6 24.7 31 1417985_at Nrarp 24.1 32 1418690_at Ptprz1 24.0 33 1443745_s_at Dmp1 23.3 34 1416211_a_at Ptn 23.2 35 1440487_at Dcc 22.8 36 1448978_at Ngef 22.8 37 1417151_a_at Ntsr2 22.7 38 1457800_at 2900019G14Rik 22.7 39 1435094_at Kcnj16 21.8 40 1427482_a_at Car8 21.7 41 1448110_at Sema4a 21.4 42 1429647_at 1700027L20Rik 20.8 43 1435417_at AI464131 20.7 44 1455462_at Adcy2 20.2 45 1458492_x_at Hnt 20.2 46 1429022_at Adcyap1r1 19.7 47 1449388_at Thbs4 19.7 48 1424208_at Ptger4 19.1 49 1435487_at Grid2 19.1 50 1435895_at D930023J12Rik 19.0 51 1455636_at Lsamp 18.6 52 1422823_at Eps8 18.3 53 1423343_at Slco1c1 18.1 54 1428866_at 2810037O22Rik 17.6 55 1434430_s_at Adora2b 17.5 56 1434141_at Gucy1a3 17.4 57 1445676_at Kcnn2 17.2 58 1434742_s_at Aifm3 17.0 59 1418310_a_at Rlbp1 16.8 60 1448602_at Pygm 16.6 61 1433965_at Atp8a1 16.1 62 1425408_a_at 2610034M16Rik 16.0 63 1457495_at 2900052N01Rik 16.0 64 1435936_at Slc13a5 15.9 65 1419028_at Arpp21 15.9 66 1434136_at 6332401O19Rik 15.6 67 1438431_at Abcd2 15.5 68 1416718_at Bcan 15.4 69 1422733_at Fjx1 15.1 70 1419383_at S100b 15.0 71 1435308_at Fut9 14.9 72 1434735_at Hlf 14.8 73 1450713_at Cspg5 14.7 74 1457743_at Rgs7bp 14.7 75 1436575_at Grin3a 14.4 76 1450468_at Myoc 14.4 77 1460444_at Arrb1 14.2 78 1436698_x_at BC054438 14.1 79 1450683_at Tagln3 14.0 80 1439048_at 2900078E11Rik 13.9

Page 56 of 58 Supplemental Table S21: Genes statistically enriched in cultured astroglia compared to in vivo astrocytes. Probe Set ID Gene Name Fold Enriched In Cultured Astroglia Over In Vivo Astrocytes 1 1423062_at Igfbp3 101.8 2 1418979_at Akr1c14 88.0 3 1449254_at Spp1 74.5 4 1460330_at Anxa3 61.6 5 1448213_at Anxa1 54.9 6 1450876_at Cfh 52.8 7 1419663_at Ogn 48.8 8 1416164_at Fbln5 43.8 9 1460049_s_at 1500015O10Rik 42.6 10 1423281_at Stmn2 39.6 11 1456174_x_at Ndrg1 38.4 12 1421811_at Thbs1 35.6 13 1448735_at Cp 34.5 14 1422629_s_at Shroom3 33.1 15 1448314_at Cdc2a 30.4 16 1448182_a_at Cd24a 28.6 17 1431057_a_at Prss23 27.4 18 1420760_s_at Ndrl 26.8 19 1416121_at Lox 25.6 20 1450981_at Cnn2 25.2 21 1424885_at Klhdc8a 24.5 22 1424254_at Ifitm1 23.4 23 1433966_x_at Asns 23.3 24 1454694_a_at Top2a 23.2 25 1455978_a_at Matn2 22.8 26 1456084_x_at Fmod 22.6 27 1425964_x_at Hspb1 22.4 28 1452242_at Cep55 20.6 29 1437409_s_at Gpr126 20.1 30 1455627_at Col8a1 19.7 31 1448326_a_at Crabp1 19.7 32 1451246_s_at Aurkb 19.5 33 1419706_a_at Akap12 19.1 34 1418572_x_at Tnfrsf12a 19.0 35 1449340_at Sostdc1 18.6 36 1416529_at Emp1 18.5 37 1448627_s_at Pbk 18.5 38 1460351_at S100a11 17.2 39 1418476_at Crlf1 17.1 40 1429987_at 9930013L23Rik 17.1 41 1423505_at Tagln 16.8 42 1422598_at Casq1 16.7 43 1419091_a_at Anxa2 16.4 44 1451791_at Tfpi 16.0 45 1418778_at Ccdc109b 15.9 46 1455900_x_at Tgm2 15.9 47 1439764_s_at Igf2bp2 15.7 48 1434447_at AI838057 15.7 49 1427883_a_at Col3a1 15.6 50 1419573_a_at Lgals1 15.5 51 1448613_at Ecm1 15.3 52 1419513_a_at Ect2 15.0 53 1425175_at C1ql3 14.6 54 1448232_x_at Tuba1c 14.5 55 1425458_a_at Grb10 14.5 56 1450759_at Bmp6 14.2 57 1422437_at Col5a2 14.2 58 1425016_at Ephb2 14.1 59 1452954_at Ube2c 14.1 60 1435917_at Ociad2 14.0 61 1429348_at Sema3c 13.7 62 1439364_a_at Mmp2 13.7 63 1438684_at Nuak1 13.7 64 1435184_at Npr3 13.7 65 1417457_at Cks2 13.6 66 1455239_at 6330512M04Rik 13.4 67 1418203_at Pmaip1 13.2 68 1448205_at Ccnb1-rs1 13.2 69 1460603_at Samd9l 13.2 70 1445897_s_at Ifi35 13.1 71 1437846_x_at Bace2 12.9 72 1416558_at Melk 12.9 73 1417419_at Ccnd1 12.7 74 1437463_x_at Tgfbi 12.6 75 1417262_at Ptgs2 12.6 76 1420377_at St8sia2 12.6 77 1450920_at Ccnb2 12.6 78 1419153_at 2810417H13Rik 12.4 79 1434748_at Ckap2 12.4 80 1455980_a_at Gas2l3 12.2

Page 57 of 58 Supplemental Table S22: Genes in the Notch Signaling pathway Probe Set ID Cultured Astrocytes Astrocytes Neurons OPCs Myelin Gene Symbol Gene Title Astroglia P7 P17 P16 Oligos

1421858_at 1052 3214 2451 171 2422 2484 Adam17 a disintegrin and metallopeptidase domain 17 1424979_at 310 544 647 220 403 365 Aph1a anterior pharynx defective 1a homolog (C. elegans) 1435793_at 498 810 1370 516 1382 434 Aph1b anterior pharynx defective 1b homolog (C. elegans) 1425822_a_at 2257 3787 6819 3292 3413 627 Dtx1 deltex 1 homolog (Drosophila) 1439429_x_at 518 555 661 292 1549 575 Dtx2 deltex 2 homolog (Drosophila) 1420752_at 681 960 2115 671 1184 324 Dtx3 deltex 3 homolog (Drosophila) 1436545_at 1779 2023 1202 1229 1311 260 Dtx4 deltex 4 homolog (Drosophila) 1450779_at 24471 32286 27467 203 19144 1932 Fabp7 fatty acid binding protein 7, brain 1450728_at 685 7510 10392 1056 411 149 Fjx1 four jointed box 1 (Drosophila) 1418102_at 894 4237 3253 477 486 17 Hes1 hairy and enhancer of split 1 (Drosophila) 1456010_x_at 37 15031 10385 60 6885 77 Hes5 hairy and enhancer of split 5 (Drosophila) 1415999_at 4335 4758 5686 1247 2965 457 Hey1 hairy/enhancer-of-split related with YRPW motif 1 1418106_at 59 605 311 13 223 20 Hey2 hairy/enhancer-of-split related with YRPW motif 2 1420643_at 203 468 356 46 41 26 Lfng lunatic fringe gene homolog (Drosophila) 1418634_at 3933 5909 6832 147 5033 869 Notch1 Notch gene homolog 1 (Drosophila) 1455556_at 2351 4570 3060 234 170 352 Notch2 Notch gene homolog 2 (Drosophila) 1421965_s_at 396 2206 1480 118 66 106 Notch3 Notch gene homolog 3 (Drosophila) 1417985_at 429 9740 12607 352 5302 277 Nrarp Notch-regulated repeat protein 1416891_at 2118 1447 2206 1587 2810 720 Numb numb gene homolog (Drosophila) 1454896_at 3248 1411 1843 724 2161 3386 Rbpsuh recombining binding protein suppressor of hairless (Drosophila)

Notch ligands 1419204_at 236 396 180 320 3525 580 Dll1 delta-like 1 (Drosophila) 1449236_at 100 218 92 194 2836 65 Dll3 delta-like 3 (Drosophila) 1421827_at 76 100 68 132 65 77 Dll4 delta-like 4 (Drosophila) 1423671_at 7403 12448 17523 11773 12091 3532 Dner delta/notch-like EGF-related receptor 1434070_at 192 319 226 24 370 68 Jag1 jagged 1 1426430_at 107 88 140 265 103 91 Jag2 jagged 2

Reference Cell Type Enriched Genes 1440142_s_at 16201 9742 13827 31 13 77 Gfap glial fibrillary acidic protein 1448768_at 12 7 14 6 14 24964 Mog myelin oligodendrocyte glycoprotein 1433884_at 315 326 339 18197 749 239 Syt1 synaptotagmin I

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