GSM3406695: HN120-Met-H3K27ac; Homo sapiens; ChIP-Seq
For chromatin prepration one million cell were cross-linked (10% FBS/DMEM containing 1.5% PFA) for 30 min. Followed by addition of 1/20 volume of 2.5M glycine, next these cells were incubated on ice for 5 min. Crosslinked cellular material was re-dissolved in buffer contacting 10 mM Tris-HCl, pH8, 1 mM EDTA, 0.5 mM EGTA, 1X protease inhibitor cocktail (Roche) followed by sonication for 10-12 cycles (30 sec on/off). Chromatin was pulled by incubation with H3K4me3 or H3K427ac antibody-coupled dynabeads and 150 μl of ChIP buffer (433 mM NaCl, 0.43% sodium deoxycholate, 4.3% Triton X-100). The 1/10 of chromatin mixture was separated for input control. The beads were washed 5x with RIPA buffer and incubated in 100 μl of elution buffer (50 mM Tris-HCl, pH8, 1% SDS, 10 mM EDTA) at 65°C for 20 min with brief vortexing followed by recovery of immunoprecipitated chromatin. The immunoprecipitated chromatin and input samples were de-crosslinked by incubation at 65°C overnight and subsequent incubation with 0.2 μg/μl of RNase A at 37°C for 1 hr and with 0.2 μg/μl of proteinase K at 55°C for 2 hr. The DNA was extracted by phenol/chloroform/isoamylalcohol (49:49:2) and ethanol precipitated in presence of 40 μg of glycogen. After washing with 70% ethanol, pellets were resuspended in 10 mM Tris-HCl, pH8. Multiplexed ChIP-seq libraries were generated using NEBNext ChIP-seq library Prep kit (NEB) following the instructions from the manufacturer. The sequencing was performed on Illumina HiSeq-2500. We employed Dfilter to call ChIP peaks in the naïve, drug-resistant and drug-holiday H3K4me3 and H3K27ac ChIP-seq libraries relative to input controls.