Chromatin immunoprecipitation in CDK8-KO and CDK8/HDAC3-KO HAP1 cells

ChIP was performed in biological quadruplicates.10 million HAP1 cells were plated in 15-cm dishes 24 h prior to harvesting. Cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with 0.125 M Glycine for 5 min. After two washes with ice-cold PBS, cells were scraped and collected. Cell pellets were resuspended in 1 mL cytoplasmic buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25 % Triton) and incubated on ice for 10 min. Nuclei were pelleted by centrifugation at 1500 x g for 5 min, washed once with the same buffer and lysed with nuclei lysis buffer (50 mM Tris pH 8.0, 5 mM EDTA, 1% SDS) and incubated for 10 min on ice. Chromatin was sheared using a Bioruptor Plus sonicator (Diagenode Cat. No. B01020001) at high power for 16 cycles (30 sec on, 30 sec off). DNA fragment size (300-400 bp) was confirmed on a 2% agarose gel. Sonicated chromatin was cleared by centrifugation at 20,000 x g for 10 min at 4°C and diluted 1:10 with ChIP equilibration buffer (16.7 mM Tris pH 8.0, 1.67 mM EDTA, 167 mM NaCl, 1.1% Triton). Chromatin was pre-cleared with Protein G Dynabeads (Invitrogen) for 1 h at 4°C with rotation. For immunoprecipitation, 3 µg of RNAPII antibody were bound to 30 µL of Protein G Dynabeads in PBS at 4°C for 2 h with rotation. Beads were washed twice with PBS, resuspended in RIPA buffer (20 mM Tris pH 8.0, 2 mM EDTA, 0.1% SDS, 1% Triton, 150 mM NaCl) and incubated with pre-cleared chromatin overnight at 4°C with rotation. Beads were washed twice with 1 mL RIPA buffer, then once with 1 mL high salt RIPA buffer (500 mM NaCl), 1 mL LiCl buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP40), and finally twice with 1 mL TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0). Beads were resuspended with 100 µL nuclear lysis buffer, and 10 % input samples were diluted to 1% final SDS concentration. All samples were incubated at 70°C overnight to reverse crosslink Samples were then treated with 5 µL Proteinase K (20 mg/mL) at 60°C for 30 min, followed by RNaseA treatment (0.2 mg/mL) for 2 h at 37°C. DNA was purified twice with phenol-chloroform-isoamyl alcohol extraction, and precipitated with ethanol and sodium acetate. Purified DNA was used for library preparation with the ThruPLEX DNA-seq Kit (Takara, Cat. No. R400674) according to the manufacturer's instructions. Libraries were sequenced on two lanes of 1.5B PE50 using an illumina NovaseqX sequencer.