GSM5516200: H3K4me2_hbdTSC_E2_rep2; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation (ChIP) assay was performed as previously described (Mendenhall et al., 2013). Briefly, cells from two biological replicates per line were fixed for 10min at RT with a final formaldehyde concentration of 0.8%. Formaldehyde was quenched with glycine at a final concentration of 125mM. The cells were then lysed with lysis buffer (100mM Tris-HCl, 300mM NaCl, 2% Triton X-100, 0.2%v sodium deoxycholate and 10mM Cacl2) supplemented with EDTA-free protease inhibitor (Roche, 11873580001) for 20min on ice. The chromatin was digested by adding MNase (Thermo Scientific, 88216) for 20min at 37C and MNase was inactivated by adding 20mM EGTA. The fragmented chromatin was added to pre-bounded Dynabeads (A and G mix, Invitrogen, 10004D/ 10002D) using H3K4me2 antibody (Millipore, 07-030) at 2 ug per reaction. 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) and twice with 10mM Tris-HCl pH = 8. DNA was purified by adding RNase A (Thermo Scientific, EN0531) and incubated for 30 min at 37C and then with Proteinase K (Invitrogen, 25530049) for 2h. The DNA was eluted by adding 2X concentrated elution buffer (10mM Tris-HCl, 300mM NaCl, 1% SDS, 2mM EDTA) and reverse crosslinked overnight at 65C. The DNA was then extracted using AMPure XP beads (Beckman Coulter Genomics, A63881). ChIP sample libraries were prepared according to Illumina Genomic DNA protocol.