title: GSM3071296: A1A3_Brg1KD_EtOH_Input_ChIPseq; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-seq, cells were fixed with 1% formaldehyde at 37C for 10 minutes for all targets except BRG1, for which cells were fixed for 20 minutes. After quenching with glycine, cell pellets were washed Hypotonic buffer (10mM HEPES-NaOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10% glycerol, 0.1% Triton X-100, and HALT protease inhibitors (ThermoFisher)) and resuspended in Shearing buffer (10 mM Tris-HCl pH 8.0, 1mM EDTA, 0.5mM EGTA, 0.5mM PMSF, 5mM Sodium Butyrate, 0.1% SDS, and HALT protease inhibitors (ThermoFisher) and chromatin was fragmented by sonication with the Covaris S220. Chromatin was diluted two-fold in 2xIP buffer (20 mM Tris-HCl pH 8.0, 300mM NaCl, 2mM EDTA, 20% Glycerol, 1% Triton X-100, 0.5mM PMSF, 5mM Sodium Butyrate, and HALT protease inhibitors (ThermoFisher)) and immunoprecipitation was performed with 1ug antibody per 100ug chromatin. Immune complexes were captured using protein A and G dynabeads, washed once each with low salt (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.1 % SDS), high salt (same as low salt buffer, except 500 mM NaCl), and LiCl buffer (Tris-HCl pH 8.0, 250 mM LiCl, 2 mM EDTA, 1 % NP-40, 1 % (wt/vol) sodium deoxycholate), and twice with TE. Eluted DNA was RNaseA and Proteinase K  treated and purified using Qiagen PCR purification columns. For RNA-seq, RNA was isolated from treated A1-2 and A1A3 cells using Qiagen RNeasy kits with on-column DNase treatment. ChIP-seq libraries were generated using the Illumina Nextara-XT library generation kit, RNA-seq libraries were generated at the National Intramural Sequencing Center using Ribo-Zero Gold
attributes.tag: GEO Accession
attributes.value: GSM3071296