H3k9ac antibody

Cells were fixed in 1% formaldehyde for 10 min at room temperature. The crosslinking reaction was quenched with 0.125 M glycine. After washing with PBS, cells were lysed with lysis buffer (50 mM Tris·HCl pH 8.0, 2 mM EDTA, 15 mM NaCl, 1% SDS, 0.5% deoxycholate, protease inhibitor cocktail, 1 mM PMSF), followed by sonication and centrifugation. The supernatant was collected and precleared in dilution buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100) with protein A/G Sepharose and pre-treated salmon DNA. The precleared samples were incubated overnight with H3K9ac antibody (2 μg/sample, Millipore) or appropriate control IgGs (Santa Cruz), and protein A/G Sepharose (Invitrogen). After washing sequentially with a series of buffers, the beads were heated at 65°C to reverse the crosslink. DNA fragments were purified and analyzed. Real-time PCR was performed with primers as described (Zhong et al., 2010 (link)):
LDHB-ChIP-5’: AGAGAGAGCGCTTCGCATAGLDHB-ChIP-3’: GGCTGGATGAGACAAAGAGCALDOC-ChIP-5’: AAGTGGGGCACTGTTAGGTGALDOC-ChIP-3’: GTTGGGGATTAAGCCTGGTTPFKM-ChIP-5’: TTAAGACAAAGCCTGGCACAPFKM-ChIP-3’: CAACCACAGCAATTGACCACLDHA-ChIP-5’: AGGGGGTGTGTGAAAACAAGLDHA-ChIP-3’: ATGGCTTGCCAGCTTACATCLDHA-ChIP-1Kb-5’: TGCAAGACAAGTGTCCCTGTLDHA-ChIP-1Kb-3’: GAGGGAATGAAGCTCACAGC