GSM7884776
Crude nuclear pellets from macrophages were collected by centrifugation, resuspended in lysis buffer, and incubated on ice for 10 minutes. The chromatin was sheared by sonication at 4°C using a Bioruptor 300 at the highest setting for fifteen 1-minute cycles of 30 seconds on and 30 seconds off to generate chromatin fragments of approximately 200-400 bp in length. The soluble chromatin was diluted 1:10 with dilution buffer and precleared with protein A agarose beads. The precleared supernatant was incubated overnight at 4°C with antibodies against H3K27ac. After washing, the immunoprecipitated material was eluted by adding 100 mM NaCl and incubating at 65°C for 12 hours. After removing protein and RNA, the enriched genomic DNA was extracted with phenol:chloroform:isoamyl alcohol followed by ethanol precipitation. The immunoprecipitated DNA was dissolved in water and analysed by ChIP-seq.