GSM4455978: RS4.11 DMSO H3K27ac 10h ChIP-seq 1; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1/10 of volume of 11% formaldehyde solution (37% formaldehyde (Sigma-Aldrich), 5 M NaCl, 0.5 M EDTA pH 8.0, 0.5 M EGTA, 1 M HEPES) for 10 min gently rotating at 1 million/ml cell concentration in media. The reactions were quenched by adding glycine to a final concentration of 120 mM, and cells were washed twice with ice-cold PBS. Crosslinked lysate was flash frozen with liquid nitrogen and stored at - 80°C. Lysates were thawed, nuclei were extracted, and MNase  treated. 5 U of MNase (Thermo Fisher Scientific) was used and supernatant was diluted with dilution buffer 2.5 times. Samples were pre-cleared with 25 μl per sample Protein G Dynabeads (Thermo Fisher Scientific) for 1h at 4°C on a rotating platform. Subsequently,  input samples were collected. Next, 5 μg of the RUNX1 antibody (cat# ab23980, Abcam) for 10-16 million cells or 2.5 μg of H3K27ac antibody (cat# ab4729, Abcam) for 5-9 million cells were added. Bound chromatin fragments were collected by adding 25 μl of Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C rotating. Beads were washed with 1ml of cold wash buffers for 3 minutes. Beads were eluted twice in elution buffer (1% SDS, 100 mM NaHCO3). Immunoprecipitated chromatin was reverse crosslinked by adding NaCl to a final concentration of 0.2 M. Next, 2,65 mg/ml of RNAse A (Invitrogen) was added to each sample and crosslinks were reversed overnight at 65°C. Proteinase K treatment was performed by adding 31 μl of buffer containing 161 mM EDTA, 645 mM Tris pH 7.4, Proteinase K 0.65 mg/ml (Thermo Fisher Scientific) for 1 h at 50°C. For DNA purification, ChIP DNA Clean & Concentrator (Zymo Research, Irvine, CA, USA) was used according to manufacture´s protocol. ChIP-Seq libraries were prepared  Sequencing libraries were prepared from collected DNA by blunting, A-tailing, adaptor ligation using barcoded adapters (NextFlex, Bioo Scientific) with 13-14 cycles in PCR amplification and size selected for 225 bp-350 bp fragments by gel extraction.