title: GSM5144722: KPC_ChIPseq_Arid1a_shSmarcd3; Mus musculus; ChIP-Seq
library_construction_protocol: For RNA-seq, total RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research). For SWI/SNF subunit ChIP-seq, 6-7e6 cells were collected per condition and cross-linked first in 3mM disuccinimidyl glutarate (DSG) then in 1% formaldehyde. For histone modification ChIP-seq, 2e6 cells were collected per condition and cross-linked with 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 E229 sonicator for 10 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 Smarca4, Arid1a, H3K4me, H4K4me3 and H3K27ac then 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. ChIP-Seq libraries were generated using the NuGen Ovation Ultralow Library System V2 following the manufacturer's instructions. RNA-Seq libraries were prepared using Illumina TruSeq RNA Library Prep Kit v2 following the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM5144722