alias: GSM5075996
title: GSM5075996: HCT116-NUP93-mAC,ChIP-Seq,H3K27me3,UT; Homo sapiens; ChIP-Seq
library_construction_protocol: HCT116 cells were crosslinked in 2 mM discuccinimidyl glutarate (DSG, CovaChem, 13301-100) for 30 min first, then in 1% formaldehyde for 15 min at room temperature. Except for NUP93, all other ChIP-Seq experiments used cells that were crosslinked by 1% formaldehyde for 10 min at room temperature. After quenching with 0.125M glycine, we collected cell pellets, and extracted the nuclei by using buffer LB1 [50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA (pH 8.0), 10% (v/v) glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1×cocktail protease inhibitor], and then LB2 [10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 1×cocktail protease inhibitor]. After centrifuge, cell nuclei were suspended in buffer LB3 [10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 0.1% Na-deoxycholate, 0.5% N-lauroyl sarcosine and 1×cocktail protease inhibitor], and fragmented using Q800R3 sonicator (QSONICA).Sheared chromatins were collected by centrifugation, and were incubated with appropriate antibodies at 4°C overnight. The next morning, the antibody-protein-chromatin complex was retrieved by adding 40ul Protein G Dynabeads (Thermo Fisher Scientific, 10004D). Immunoprecipitated DNA was de-crosslinked by 65ºC heating overnight, treated by proteinase-K, and were harvested by phenol chloroform or by Qiagen Quick DNA extraction kit. ChIP-Seq  library construction using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB, E7645L).
identifiers.namespace: GEO
identifiers.id: GSM5075996
attributes.tag: GEO Accession
attributes.value: GSM5075996