GSM6914676: EC, MEF2C ChIP, A , bio rep 1; Homo sapiens; ChIP-Seq
These ECs were cross-linked in 1% formaldehyde for 10 min at room temperature and the reaction was stopped with 0.125 M glycine. Cross-linked cells were lysed with ChIP lysis buffer (5 mM PIPES pH8, 85 mM KCl, 1% Igepal) with a protease inhibitor (PI) cocktail (Millipore Sigma, #11697498001). Nuclei were collected by centrifugation at 2000 rpm for 5 min at 4 °C and lysed in nuclei lysis buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS) supplemented with the PI cocktail. Chromatin was fragmented using the Bioruptor Pico (Diagenode, Denville, NJ) and diluted with 5 volumes RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA pH8, 1% Igepal, 0.25% deoxycholic acid). ChIP enrichment was performed by incubation with 3 µg MEF2C antibody (Abcam, #ab211493) or 2 µg normal rabbit IgG (ab46540, Abcam) for 16 h at 4 °C. Immune complexes were bound to 20 µl magnetic protein A/G beads (Biorad, Hercules, CA) for 2 h at 4 °C. Beads were washed 2 × with RIPA (Thermo Fisher Scientific, #J62524.AE) and 3 × with ChIP wash buffer (100 mM Tris pH8, 500 mM LiCl, 1% deoxycholic acid). The final wash was performed in ChIP wash buffer with 150 mM NaCl. Cross-links were then reversed by heating beads in 100 µl ChIP elution buffer (50 mM NaHCO3, 1% SDS) overnight at 65 °C. DNA was purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Libraries were prepared by Novogene.