GSM5444549: HepaRG d30DMSO H3K27ac Rep2; Homo sapiens; ChIP-Seq
Detached HepaRG cells were fixed with 1% formaldehyde in William's E medium for 10 min. Crosslinking was stopped by adding glycine to a final concentration of 125 mM for 5 min at room temperature. If not mentioned otherwise samples were thereafter kept on ice or at 4 °C and centrifugations were performed at 800 x g for 5 min. After washing twice with 125 mM glycine in PBS and once in PBS, cells were resuspended consecutively in 5 ml buffer A (10 mM HEPES-NaOH, 10 mM EDTA, 0.5 mM EGTA, 0.25 % Triton X-100, 1 x PIC (Complete Protease Inhibitor Cocktail Tablets)), respectively buffer B (10 mM HEPES-NaOH, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01 % Triton X-100, 1 x PIC) and rotated for 10 min followed by centrifugation each. Cells were resuspended in 600 μl buffer C (25 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.25 % SDS, 1 x PIC). Sonication of chromatin into fragments between 100 and 800 bp was performed using the Bioruptor NGS (Diagenode, Liège, Belgium) on high power setting in 200 μl volumes, either in 0.5 ml shearing tubes with 30 cycles or in 1.5 ml microtubes (Diagenode, Liège, Belgium) with 10 cycles and each cycle consisting of 30 seconds sonication and 30 seconds recovery time. Samples were diluted with double volume dilution buffer (25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.75 % Glycerol). For each immunoprecipitation 10 µl protein G beads (Invitrogen, Carlsbad, USA) were combined either with 2 μg H3K4me3 (Diagenode, Liège, Belgium, pAb-003-050), H3K27ac (pAb-196-050) or H3K4me1 (pAb-194-050) antibody, 5 μl BSA (NEB, Frankfurt am Main, Germany, 10 mg/ml) and rotated for 2 h. Then, 300 μl sonicated chromatin (equivalent to 1 million cells) were added. Immunoprecipitations of H3K27ac were supplemented with Na-Butyrate to a final concentration of 10 mM. After rotation overnight beads were consecutively washed with 1 ml of WB1 (20 mM Tris-HCl, (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.25 % SDS), twice WB2 (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.25 % SDS), WB3 (10 mM Tris-HCl, (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5 % Nonident P-40, 0.5 % Sodiumdesoxycholate) and twice TE / NaCl (10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA) under rotation for 10 min. Elution was performed using 200 µl EB (100 mM NaHCO3, 1 % SDS) for 30 min in a shaking device at room temperature. Eluates were supplemented with crosslink reversal solution (4 μl 5 M NaCl, 2 μl 0.5 M EDTA and 2.5 μl Proteinase K (20 mg/ml)) and incubated overnight at 55 °C. ChIP DNA was purified by phenol chloroform extraction followed by ethanol precipitation. Library preparation was performed using the Truseq ChIP Sample Preparation Kit (Illumina, San Diego, USA) following the manual except for omitting size selection. All ChIP-seq libraries were sequenced 2 x 100 bp paired-end on a HiSeq2500 (Illumina, San Diego, USA).