GSM6944034: Input_replicate_2; Homo sapiens; ChIP-Seq
Cells were cross-linked for 10 min by adding formaldehyde (Sigma F8775) to a final concentration of 0.8%, and then quenched for 10 min by adding glycine to a final concentration of 200 mM.  Cells were then washed, scraped, and collected into a 50-mL falcon tube (Corning 430828), and centrifuged at 1500g for 10 min. Cell pellet was lysed in 1 mL of ChIP lysis buffer (50 mM Tris-HCl at pH 8.0, 10 mM EDTA, 10 mM sodium butyrate, 1% SDS, 4× PIC [Roche 05056489001]) and passed through a 100-μm cell strainer before being sonicated for 15 min in a Covaris S220 (peak incident = 145 W, duty factor = 5%, cycle/burst = 200) until 200- to 400-bp fragments were obtained (assessed by 1% agarose gel). The sonicated chromatin was cleared by centrifugation at 13,000g for 10 min and the supernatant was diluted eightfold in ChIP dilution buffer (16.7 mM Tris at pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1% Triton X-100, 0.01% SDS) before 12 μg of anti-HA antibody (Roche 11666606001) was added, and chromatin was rotated in a 50-mL falcon tube overnight. In parallel, 60 μL of Dynabeads protein G (Invitrogen, 10004D) was washed, resuspended in ChIP dilution buffer, and blocked in 0.5 mg/mL BSA overnight at 4°C. Immunoprecipitation was carried out using blocked Dynabeads for 1 h at 4°C. The beads were washed three times each in low-salt wash buffer (20 mM Tris-HCl at pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high-salt wash buffer (20 mM Tris-HCl at pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), and LiCl wash buffer (10 mM Tris-HCl at pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% sodium deoxycholate, 1% NP-40) with beads transferred to a new DNA LoBind tube (Eppendorf Z666548) with each wash. The beads were eluted in 200 µL of elution buffer (100 mM NaHCO3, 1% SDS). Reversal of cross-linking of chromatin-immunoprecipitated DNA was done overnight at 55°C with addition of NaCl (final concentration 300 mM), 20 µg of RNase A, and 20 µg of Proteinase. Chromatin-immunoprecipitated DNA was recovered using a QIAquick PCR purification kit (Qiagen 28106). Samples library were prepped and subjected to 150-bp paired-end sequencing using a NovaSeq6000 (Illumina) at the Wellcome Trust Genomic Service, Oxford.