GSM1847316: LP1_DMSO_input; Homo sapiens; ChIP-Seq
Cells were fixed with a final concentration of 1% formaldehyde for 10 minutes at room temperature. Glycine was added to a final concentration of 0.125 M to stop crosslinking. Cells were washed twice in cold PBS followed by lysis at 4°C for 1 hour in buffer containing 10mM Tris-HCl pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.2% NP-40, + protease inhibitor cocktail (Sigma). Following lysis, nuclei were recovered by centrifugation, and resuspended in buffer containing 10 mM Tris-HCl, 0.1 mM EDTA, 5 mM MgAc2, 25% glycerol. An equal part 2X MNase buffer was added, containing 50 mM KCl, 8 mM MgCl2, 2 mM CaCl2, 100 mM Tris-HCl. Micrococcal nuclease (Roche) was added to 300 U/mL and chromatin was digested at room temperature for 20 minutes. Dilution buffer (0.1% SDS, 1.1% Triton-X 100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl) was added and nuclei were broken down by sonication. Chromatin was cleared by centrifugation and pre-cleared with protein A-conjugated magnetic beads (Life Technologies). 10-20 µg precleared chromatin was combined with 10 µg anti-EP300 antibody (Santa Cruz sc-585X) or 2.5 ug of anti-H3K18ac (Cell Signaling, 9675) or anti-H3K27ac (Abcam, ab4729) conjugated to protein A magnetic beads. DNA was purified with the MinElute kit (QIAGEN), and libraries were prepared using the Ovation Ultralow DR Multiplex System (NuGEN) according to the manufacturer’s recommendations.
GEO
GSM1847316