EC_158_HRAEC ChIP-seq H3K27ac
Two million ECs were plated on a 15-cm culture plate and cultured until confluency. The cells were crosslinked for 10 minutes using 1% paraformaldehyde. After quenching using 0.2 M glycine, cells were collected using a scraper, resuspended in SDS lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 1% SDS, 1 mM EDTA; pH 8.0) and fragmented by sonication (Branson, Danbury, CT, USA; 10 minutes). Samples were stored at -80C before use. To perform ChIP, antibodies against histone modifications (CMA304 and CMA309 for H3K4me3 and H3K27ac, respectively; (Kimura et al. 2008) were used in combination with Protein G Sepharose beads (GE Healthcare Bio-Sciences AB, Sweden). The prepared DNA was quantified using Qubit (Life Technologies/Thermo Fisher Scientific) and more than 10 ng of DNA was processed.