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Supporting Information Supporting Information Wakabayashi et al. 10.1073/pnas.1521754113 SI Materials and Methods DNA Transfection and Puromycin Selection. K562 cells were cotrans- Plasmid Preparation. Short guide RNA (sgRNA) sequences (Table fected with 1 μg total of Cas9 nuclease and sgRNA plasmids using S1) were cloned into the pSg1 vector (Addgene) and the XPR5 Lipofectamine LTX Plus Reagent (Thermo Fisher Scientific) at a lentiviral vector (Broad Institute), respectively. [The XPR5 1:2 ratio of Cas9 to sgRNA. For a control, K562 cells were co- vector contains the Cas9 nuclease and a red fluorescent protein transfectedwith1μg total of Cas9 nuclease and pLKO.1-GFP (RFP) cassette.] The Cas9 nuclease expression vector used was plasmid at a 1:2 ratio of Cas9 to pLKO.1-GFP. At 24 h after co- μ pxPR_BRD001, which contains a puromycin resistance cassette transfection, puromycin was added at a concentration of 2 g/mL, μ as a selection marker. Off-target scores for each guide were followed 24 h later by a reduction to 1 g/mL for an additional 24 h. calculated using the CRISPR design tool (CRISPR Design; Selection efficiency was assessed by flow cytometry with propidium crispr.mit.edu); only guides with a score >50 (except for a score iodide staining (to assess viability) on a FACSCanto II flow cy- of 49 in one case) were used. tometer (BD Biosciences). Limiting dilutions were performed to obtain single cell-derived clonal populations for both cells targeted Cell Culture and Lentivirus Production. The K562 cells (American with sgRNAs as well as for GFP controls. Unless specified other- Type Culture Collection) were maintained in RPMI medium 1640 wise, three matching clonal GFP controls were analyzed for each plus L-glutamine (Life Technologies) supplemented with 10% experiment. (vol/vol) FBS (Atlanta Biologicals) and 1% penicillin-strepto- mycin (PS; Life Technologies). Cells were incubated at 37 °C Identification of Deletions. Genomic DNA was isolated from single clones using the DNEasy Blood & Tissue Kit (Qiagen). PCR with 5% CO2 in air atmosphere. The 293T cells were maintained in DMEM, high glucose (Life Technologies) supplemented with analyses were performed using Platinum PCR Supermix (In- 10% FBS and 1% PS. For lentivirus production, 293T cells were vitrogen) and a T100 Thermal Cycler (Bio-Rad) with primer transfected with sgRNA constructs (Table S1) along with the pairs flanking the sgRNA target sequences, as well as with VSV-G envelope and the pDelta8.9 packaging vector using Fu- primer pairs that select for clones containing a deletion within gene6 (Roche) transfection reagent according to the manufac- the targeted GATA1 binding site (Fig. S3). Genomic DNA turer’s instructions. The next day, the medium was changed to isolated for screening was PCR-amplified using Platinum PCR Supermix (Invitrogen) on a T100 Thermal Cycler (Bio-Rad) Iscove’s modified Dulbecco’s medium (IMDM; Life Technolo- (Table S1), and purified with the Qiaquick PCR Purification Kit gies) supplemented with 2% (vol/vol) human AB plasma, 3% (Qiagen). The product was Sanger-sequenced, and trace files (vol/vol) human AB serum (Atlanta Biologicals), and 1% PS. were analyzed on FinchTV (Geospiza). Chromatograms were The next day (48 h posttransfection), viral supernatant was col- compared with DNA sequences obtained from the University of lected and filtered through a 0.45-μm filter, and then concen- California Santa Cruz (UCSC) Genome Browser (reference ge- trated by centrifuging at 68,320 × g for 2 h at 4 °C with an nome GRCH37/hg19). Optima L-100 XP ultracentrifuge (Beckman Coulter). Concen- When identifying deletions, it is important to keep in mind that trated virus was dissolved in 2 mL of supernatant overnight at K562 cells are aneuploid in certain regions. Here we aimed to 4 °C, and infection was performed the next day. completely disrupt the targeted RE regardless of the copy number + at a particular locus. For all clonal deletions (either homozygous Primary Cell Culture and Lentiviral Infection. Adult CD34 HSPCs or compound heterozygous deletions), we observed Sanger- from mobilized peripheral blood mononuclear cells (Harvard sequencing traces only with a deletion and did not observe any Stem Cell Institute Flow Cytometry Facility) were maintained in traces of the reference K562 DNA, confirming that the targeted base medium (IMDM plus 2% human AB plasma, 3% human CRE was modified in all copies. In cases with compound het- AB serum, 1% PS, 3 U/mL heparin, 10 μg/mL insulin, and erozygosity, we typically observed similar intensities for both 200 μg/mL holo-transferrin) supplemented with 3 U/mL erythro- alleles, suggesting that the loci were diploid. poietin, 10 ng/mL human recombinant stem cell factor, and 1 ng/mL – human recombinant IL-3 for days 0 6 of culture. IL-3 was Quantitative RT-PCR. RNA was extracted from selected clones – omitted on days 7 12 of culture. Cells were incubated at 37 °C using the RNEasy Plus Mini Kit (Qiagen) and the Ambion with 5% CO2. For lentiviral infection, cells (at day 2 of culture) RNAqueous–Micro Total RNA Isolation Kit (Life Technolo- × μ were spun at 931 g for 90 min at room temperature with 8 g/mL gies). cDNA was synthesized using the iScript cDNA Synthesis polybrene (Millipore), 1 mL of concentrated virus, and 1 mL Kit (Bio-Rad). Quantitative RT-PCR (qRT-PCR) was performed of IMDM supplemented with 2% human AB plasma, 3% human with iQSYBR Green Supermix (Bio-Rad) on a CFX96 Real-Time AB serum, and 1% PS. The next day, the medium was changed PCR System (Bio-Rad). Primer sequences are listed in Table S1. μ to omit the 8 g/mL polybrene and concentrated virus. At 3 d mRNA expression levels were quantified and calculated via the postinfection, RFP was measured by flow cytometry analysis ΔΔCt method and normalized to β-actin levels (55). with a BD Acuri C6 flow cytometer (BD Biosciences). Heme Quantification. A total of 250,000 cells from the controls and Fluorescence-Activated Cell Sorting and Analysis. For fluorescence- ALAS2 clones were counted with a hemacytometer and har- activated cell sorting (FACS), cells were harvested at day 10 of vested for heme quantification using the QuantiChrom Heme culture, washed with FACS buffer [3% (vol/vol) FBS in PBS], and Assay Kit (BioAssay Systems; DIHM-250). The assay was per- then resuspended in 600 μL of FACS buffer. RFP-positive cells formed in triplicate, following the manufacturer’s protocol. were then sorted using a FACSAria II cell sorter (BD Biosci- ences). For flow cytometry analysis of differentiation, cells were Porphyrin Quantification. A total of 100,000 cells from the controls harvested at day 12 of culture, washed with FACS buffer, stained and UROS clones were seeded in RPMI medium 1640 including with allophycocyanin-conjugated anti-human CD235a antibody L-glutamine supplemented with 10% FBS and 1% PS, along with (17-9987; eBioscience) and analyzed with a BD Acuri C6 flow 1mMδ-aminolevulinic acid (Sigma-Aldrich), at a density of cytometer. 50,000 cells/mL. Cells were incubated at 37 °C with 5% CO2, Wakabayashi et al. www.pnas.org/cgi/content/short/1521754113 1of12 protected from light, for 72 h. The cells were then centrifuged at experiment, and error bars represent the SEM. Disruptions of 456 × g at for 5 min at 4 °C, and 1 mL of supernatant was col- GATA1 binding motifs for selected mutations were initially lected for analysis. Samples were deproteinized with an equal identified using TRAP (57). ChIP-seq data for GATA1, TAL1, volume of 20% TCA/DMSO (1:1, vol/vol), incubated on ice for NFE2, and KLF1 were analyzed as described previously (32) and 15 min, and then centrifuged at 10,000 × g for 10 min. Porphyrins displayed as input-normalized reads per million. Raw ChIP-seq in the samples were separated by ultra-performance liquid data were obtained and combined from multiple studies (31, 32, chromatography (UPLC) in a ACQUITY UPLC system (Waters) 58–62). LDB1 ChIP-seq and FAIRE-seq in EBs were obtained on a BEH C18 2.1 × 100 μM column (Waters) as described from www.ncbi.nlm.nih.gov/geo/ (accession nos. GSE52637 and previously (56). GSE36985) and processed similarly (58, 63). GATA1 and NDR – Eluent A was 1 mol/L ammonium acetate acetic acid buffer, peaks were defined using MACS2 (64). DHS-seq for K562 cells pH 5.16, with 0.02% sodium azide, and eluent B was neat ace- was obtained from the Integrative Genomics Browser (IGV) tonitrile. The elution program was as follows: 2 min concave server, and IGV was used for visualization of genome-wide as- (Waters #8) gradient from 8% (vol/vol) to 70% (vol/vol) B, says. RNA-seq data across the erythroid lineage were obtained followed by 0.5 min at 70% B, and a 1.5-min reequilibration at from www.ncbi.nlm.nih.gov/geo/ (accession nos. GSE61566 and 8% B at a flow rate of 0.6 mL/min. Column temperature was GSE53983) and processed as described previously (32, 65). maintained at 65 °C. Porphyrins were detected using a Waters PhastCons scores derived from 100 vertebrates were downloaded ACQUITY UPLC fluorescence detector with 405 nm excitation, from hgdownload.cse.ucsc.edu/goldenpath/hg19/phastCons100way/ 619 nm emission, and a gain of 100. For porphyrin quantitation, and compared using the seqplots package in R/Bioconductor. the UPLC chromatograph was standardized using the URO I K-means clustering was used to derive clusters of conservation and COPRO III fluorescence standards UFS-1 and CFS-3, re- across GATA1 motifs. TF binding intensities at promoters (de- spectively (Frontier Scientific). All experiments were performed ± in triplicate. fined as 1 kb from the TSS) were determined using the UCSC Genome Browser tool bigWigAverageOverBed; the maximum Intracellular Flow Cytometry.
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