GSM5904691: HCT116 FOXD2 H3K27ac ChIP rep1; Homo sapiens; ChIP-Seq
Cultured cells were collected by 0.25% Trypsin-EDTA (Gibco, 25200), then washed with DPBS. Crosslinked 6.0 or 10.0 × 106 HCT116 cells for 7 min at 37 °C with 1% formaldehyde. We perforemd FOXD2-flag or 6xHis ChIP with sonicated chromatin in RIPA buffer (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% NP-40, 0.01% SDS, and 0.5% sodium deoxycholate, 1mM PMSF and 1X protease inhibitor cocktail) with 4μg of antibody; MLL4/KMT2D antibody (Santa crz, sc-293217) or Anti-6X His tag® antibody [HIS.H8] (Abcam, ab18184) or Monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich, F1804). For histone ChIP using MNase without crosslinking method, we resuspended 1.0 x 106 HCT116 cells in 50ul of nuclei isolation buffer (Sigma-Aldrich, NUC101) and incubated on ice for 10 min. We then proceeded to MNase digestion with final concentration 2 units/μl (NEB, M0247), pre-clearing and immunoprecipitated chromatin in ChIP buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1x Protease inhibitor cocktail, and 1 mM PMSF) with 2μg of antibody; H3K4me3 (Abcam, ab8580) or H3K4me1 (Abcam, ab8895) or H3K27ac (Abcam, ab4729).