GSM7978139: ChIP-seq Pol II_HCT116; Homo sapiens; ChIP-Seq
ChIP was performed as previously described. First, approximately 2× 107 HCT 116 cells were crosslinked using 1% formaldehyde (Sigma) at room temperature for 10 minutes, and cross-linking was quenched by glycine (final concentration is 125 mM) for 5 minutes. Then, cells were rinsed twice with ice-cold 1 × PBS, harvested by centrifugation, and resuspended in 10 mL of ChIP lysis buffer I (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Igepal, 0.25% Triton X-100) in the presence of protease inhibitors cocktail (Sigma) for up to 10 minutes on ice. Next, the cells were suspended in ChIP lysis buffer II (10 mM Tris-HCl [pH 8.0], 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease inhibitor) for 10 minutes at room temperature. Fixed chromatin was sonicated into 200-500 bp fragments (Sonicator, Diagenode) in ChIP lysis buffer III (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1× protease inhibitor). Chromatin extracts were incubated with specific antibody (anti-Pol II, anti-Pol II-pSer2, anti-SPT5, anti-SPT5-CTR1/PRD, anti-NELFE, anti-AFF4) and protein A agarose beads at 4°C overnight. Immunoprecipitates were washed with RIPA buffer (50 mM HEPES-KOH [pH 7.5], 500 mM LiCl, 1 mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate) for five times and TE twice. After the final wash, DNA was eluted with Elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1.0% SDS), and reverse-cross-linked at 65°C. Subsequently, the eluted DNA were treated with Protease K and RNase A, and were purified by FastPure Gel DNA Extraction Mini Kit (Vazymes). ChIP-seq libraries were prepared using VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazymes), and sequencing was performed using an Illumina NovaSeq 6000 platform