GSM5600261: H3K27Ac Clino 24h; Mus musculus; ChIP-Seq
Ten million C2C12 cells were used. Briefly, the cells were crosslinked in 1% formaldehyde, after which they were collected and resuspended for 5 min in swelling buffer (10 mM HEPES/KOH pH 7.9, 85 mM KCl, 1 mM EDTA, 0.5% IGEPAL CA-630) containing protease inhibitors. The cells were then spun down and resuspended in 500 µl of lysis buffer (50 mM Tris/HCL pH7.4, 1 % SDS, 0.5 % Empigen BB, 10 mM DETA) containing protease inhibitors, and chromatin was sheared by sonication. The lysate was diluted with 750 µl of dilution buffer (20 mM Tris/HCl, 100 mM NaCl, 0.5% TritonX-100, 2 mM EDTA), after which 1% was taken as input DNA, immunoprecipitation was carried out overnight using Dynabeads protein G (Thermo Fisher) coated with H3K27Ac antibody (Abcam, Ab4729). Thereafter, the beads were washed two times each with wash buffer I (20 mM Tris/HCl, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), wash buffer II (10 mM Tris/HCl, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Na-deoxycholate, 1 mM EDTA), TE plus 0.2% Triton X-100, and TE plus 50 mM NaCl and then eluted with elution buffer containing 1% SDS. DNA was reverse crosslinked and purified using a QIAquick PCR purification Kit (Qiagen) according to the manufacturer's instructions. ChIP-seq libraries were prepared using a NEBNext UltraII RNA Library Prep kit for Illumina (NEB) according to the manufacturer's protocol.