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1 SUPPLEMENTAL MATERIALS a Transcriptome Database For 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.
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