GSM5962442: HCT116-NUP93-mAC,ChIP-Seq for BRD4,UT, biological replicate 1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
BRD4
Cell type
Cell type Class
Digestive tract
Cell type
HCT 116
Primary Tissue
Colon
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
colorectal cancer cells
cell line
HCT116
antibody
BRD4(Cat#: A301-985A100, RRID:AB_2620184)
treatment
untreated
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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).