GSM5219265: H3K27ac_OSKM_D09_A_S5 (ChIP-seq); Mus musculus; ChIP-Seq
DNA was extracted using AMPure XP beads (Beckman Coulter Genomics, A63881). Chip sample libraries were prepared according to Illumina Genomic DNA protocol as described (Mikkelsen et al., 2007). Chromatin immunoprecipitation (ChIP) assay was performed as previously described (Mendenhall et al., 2013). Briefly, cells were fixed for 10 min at RT with a final concentration of 0.8% formaldehyde. Formaldehyde was quenched with glycine for a final concentration of 125mM. The cells were then lysate with lysis buffer (100mM Tris-HCl, 300mM NaCl, 2% Triton® X-100, 0.2%v sodium deoxycholate, 10mM Cacl2) supplemented with EDTA free protease inhibitor Roche- 11873580001 for 20 min at Ice and the chromatin was digested by MNase (micrococcal nuclease)- Thermo Scientific™- 88216 for 20 min at 37OC. MNase was inactivated by 20mM EGTA. The fragmented chromatin was incubated with pre-bounded Dynabeads (A and G mix) - Invitrogen 10004D/ 10002D using H3K27ac antibody (Abcam, ab4729) and H3K4me2 antibody (Millipore, 07-030).  Samples were then washed twice with RIPA buffer, twice with RIPA high salt buffer (NaCl 360mM), twice with LiCl wash buffer (10mM Tris-Hcl, 250mM LiCl, 0.5% DOC, 1mM EDTA, 0.5% IGEPAL), twice with 10mM Tris-HCl pH=8. DNA was purified by incubating the samples with RNAse A (Thermo Scientific™ EN0531) for 30 min at 37OC followed by a 2 hours incubation with Proteinase K (Invitrogen™ 25530049). DNA was eluted by adding 2X concentrated elution buffer (10mM Tris-HCl, 300mM NaCl, 1% SDS, 2mM EDTA) and then reverse crosslinked overnight at 65OC.