title: GSM8059246: H3K27ac, INPUT, DMSO; Homo sapiens; ChIP-Seq
library_construction_protocol: 107cells were fixed on plate with 1% paraformaldehyde for 15min and neutralized with 125mM glycine.  Fixed cells were washed twice with ice-cold PBS and scraped off in ice-cold PBS containing Protease Inhibitor Cocktail (Sigma–Aldrich #P2714). Cells were pelleted and then resuspended in ChIP Lysis Buffer (0.5% (w/v) sodium dodecyl sulfate (SDS), 8 mM EDTA, 40 mM Tris-HCl (pH 8), protease inhibitor cocktail). Cells were kept on ice for one hour before sonication at 4°C (Bioruptor, Diagenode) to an average DNA length of 200–500 bp by 15 pulses of 10 secs on and 10 secs off at high intensity. Cell debris was removed by centrifugation at top speed for 20 min and chromatin concentration recorded using OD260. Samples were diluted to a final concentration of 200mg/ml in ChIP dilution buffer (0.01% (w/v) SDS, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8), 1.1% (v/v) Triton X-100, 167 mM NaCl, protease inhibitor cocktail) and 600mg chromatin was used for each immunoprecipitation. 4 mg of anti-H3K27Ac coupled to 50 ml Pierce™ Protein A/G Magnetic Beads was added to each sample and rotated overnight at 4°C. For spike-in normalization 4 mg of drosophila chromatin (Active motif # 53083) and 2 ml spike-in antibody (Active motif # 61686) were added per sample. The beads were harvested by magnets and washed once for 10 min in the cold room by rotation with 1 ml of ChIP Low Salt Wash Buffer (0.01% (w/v) SDS, 2 mM EDTA, 20 mM Tris-HCl (pH 8), 1% (v/v) Triton X-100, 150 mM NaCl), 1 ml of ChIP High Salt Wash Buffer (0.01% (w/v) SDS, 2 mM EDTA, 20 mM Tris-HCl (pH 8), 1% (v/v) Triton X-100, 500 mM NaCl), and 1 ml of ChIP LiCl Wash Buffer (250 mM LiCl, 1% (w/v) IGEPAL, 1% (w/v) sodium deoxycholate, 10 mM Tris-HCl (pH 8), 1 mM EDTA). Finally, the beads were washed twice for 2 min in the cold room by rotation with 1 ml of TE buffer (1 mM EDTA, 10 mM Tris-HCl (pH 8)). Antibody-bound chromatin fragments were digested and crosslinks reversed with Reversal Buffer (0.0075% (w/v) SDS, 200 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl (pH 7.5), 25 µg proteinase K (Thermo Fisher Scientific) by incubating at 50°C for two hours with shaking and subsequently seven hours at 65°C. ChIP DNA samples were purified with phenol-chloroform extraction and ethanol precipitation with glycogen (Thermo Fisher Scientific) as carrier. ChIP-seq libraries were generated using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB # E7645S) following the manufacturer's protocol.