GSM8107958: R1/E cells shControl, H3K9ac; Mus musculus; ChIP-Seq
40 million cells were fixed with 1% formaldehyde for 10 minutes at room temperature. Fixation was stopped with 0.125 M glycine, the cell pellets were washed with cold PBS three times. And cells were prepared using a step-by-step process involving Lysis buffer 1 (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100), Lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), and Lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholdate, and 0.5% N-lauroylsarcosine). All buffers were freshly supplemented with  1 mM PMSF, 5 mg/mL leupeptin, 5 mg/mL pepstatin, and 10 mg/mL aprotinin. Chromatin fraction was sonicated with a Bioruptor® Plus to shear DNA to a size of 300-500bp. Samples were centrifuged at 12,000 ×g for 10 min at 4°C and supernatants collected. Specific antibodies and protein A-agarose beads were used to immunoprecipitate the antibody/protein/DNA complexes. The protein-DNA-antibody complexes were washed 5 times with cold Wash buffer(50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% Na-Deoxycholdate), and eluted in elution buffer TES (10 mM Tris-HCl+1% SDS). Crosslinks were reversed overnight at 65°C. After RNase A and proteinase K treatment, the DNA was purified by phenol/chloroform extraction and ethanol precipitated and was quantified using QubitTM 3.0. ChIPed DNA was reverse-crosslinked and purified for DNA library construction followed by sequencing. Libraries were sequenced on HiSeq PE150.