H3K27ac ChIP-seq of mus musculus (del2k): cultured mouse embryonic fibroblasts
1-5 * 106 cells were crosslinked with 1% formaldehyde at RT for 10 min. Cells were lysed on ice in 0.5 ml lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, 0.4-1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, Complete Mini EDTA-free protease inhibitor (Roche)). Lysed chromatin was fragmented using the BANDELIN SONOPULSE until reaching a fragment size of 150500 base pairs . SDS concentration and the number of sonication cycles were adjusted according to a cell type and shearing efficiency. SDS concentration was reduced before the immunoprecipitation step (~0.17% SDS final) by dilution with SDS-free lysis buffer. Chromatin was pre-cleared with Protein A magnetic beads, and 100L of the precleared solution was saved as an input control. During this time, an aliquot of Protein A beads was  rotated with the respective antibody at 4 (1.5g or 5g of antibody for CTCF and H3K27ac ChIP reaction, respectively). Protein-DNA complexes were then immunoprecipitated overnight at 4 C with rotation. The beads were washed at 4 in the following buffers: lysis buffer (0.1% SDS, twice), lysis buffer containing 0.5M NaCl (twice), LiCl buffer (0.25 M LiCl, 0.5% IGEPAL-630, 0.5% sodium deoxycholate, twice), TE (pH 8.0) plus 0.2% Triton X-100 (once), and TE (pH 8.0, once). Crosslinks were reversed at 65 overnight and DNA was purified. Sequencing libraries were prepared using the KAPA Hyper Prep kit.