GSM3476782: H3K27me3-R1159Q-ChIPseq; Homo sapiens; ChIP-Seq
For RNA-seq, RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research). For ChIP-Seq of Histone modificatons, ~1.5E6 hNSCs were collected and cross-linked in 1% formaldehyde. For ChIP-Seq of SMARCA2 and SMARCA4, ~6E6 hNSCs were first collected and cross-linked in 2mM disuccinimidyl glutarate (DSG) then in 1% formaldehyde. After quenching the excess formaldehyde with 125 mM glycine, the fixed cells were washed, pelleted and flash-frozen. Upon thawing, the cells were resuspended in lysis solution (50 mM HEPES-KOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 and incubated on ice for ten minutes. The isolated nuclei were washed with wash solution (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl) and shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 8) then sheared in a Covaris E220 sonicator for 10-20 minutes to generate DNA fragments between ~ 200-1000 bp. After clarification of insoluble material by centrifugation, the chromatin was immunoprecipitated overnight at 4C with antibodies against H3K4me, H3K4me3, H3K27me3, H3K27ac, SMARCA2 and SMARCA4 bound to Protein A+G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, 0.1% SDS). Antibody bound DNA were washed and treated with Proteinase K and RNase A and the purified ChIP DNA was used for library generation for subsequent sequencing. For ATAC-seq, ~50000 hNSCs were collected and washed with cold PBS. Cells were resuspended in resuspension buffer (10mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2) and then lysed in lysis buffer (10mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40). Nuclei was collected and incubated in transposition mix containing Tn5 transposase(Illumina). Purified DNA was then ligated with adapters to prepare library for sequencing. ChIP-Seq libraries were generated using the NuGen Ovation Ultralow Library System V2 following the manufacturer's instructions. RNA-Seq libraries were prepared using either Illumina TruSeq RNA Library Prep Kit v2 or Illumina TruSeq Stranded mRNA Kit following the manufacturer's instructions.
GEO Accession
GSM3476782