GSE125033
ChIP-Seq analysis of SMARCA4 binding sites in human NSC
We performed large-scale, pooled shRNA screens targeting 538 epigenetic regulators in genetically identical human stem cells, where each gene was targeted on avarage by 12 shRNAs. CD34+ HSPCs, used as a starting point of the study, were reprogrammed to iPSCs, which were differentiated to NSC. These cells were transduced with the pooled shRNA library and samples were collected 2 days post transduction (dpt), 12 dpt, and 22dpt - allowing 5 cell doublings. PCR amplified gDNA carrying the shRNA barcodes was sequenced on a HiSeq2500 Illumina sequencer. Analysis of NGS results, based on the change of shRNA representation (read count) and represented by z-scores, identified SMARCA4 as a differential regulator (SMARCA4 shRNAs were depleted in HSPC vs enriched in NSC sreens). We performed RNA-seq experiments as part of the characterization dataset for the isogenic HSPC, HSPC-derived iPSC, and iPSC-derived NSC. RNA samples from CD34+ HSPC were harvested in parallel to gDNA isolation on 12- and 22-days post transduction, during the RNAi screen. In addition, untreated iPSC and NSC RNA samples were sequenced as part of their characterization. RNA samples were sequenced with poly-dT enrichment using single-end sequencing. Each sample was sequenced at a depth of yielding at least 20 million reads. We performed a ChIP-seq analysis of the SMARCA4 binding sites in hNSC, which we identified in the large-scale RNAi screens as a candidate, involved in regulation of self-renewal and differentiation. The pull-down experiment was performed on the untreated NSCs. We show that SMARCA4 has a prominent binding tendency for the TSS throughout the genome, suggesting a role for SMARCA4 in transcriptional regulation. Overall design: Pooled shRNA screen on genetically identical CD34+ HSPC and NSC, RNA-Seq transcriptome profiling of isogenic CD34+ HSPC, iPSC, and NSC and examination of SMARCA4 binding sites in an iPSC-derived NSC line by ChIP-Seq.