C15410194

Antibodies used were as follows: anti-H4K5acK8ac (mouse monoclonal) [23 (link)], anti-H3K4me3 (rabbit monoclonal, 17–614, Millipore), anti-H3K4me1 (rabbit polyclonal, C15410194, Diagenode), anti-H3K27ac (rabbit polyclonal, ab4729, Abcam), and anti-BRD4 (rabbit polyclonal, A301-985A100, Bethyl Laboratories).

ChIP was performed according to standard protocol [50 (link)] with minor modifications. Paraformaldehyde (1 %) cross-linking was carried out for 10 min followed by the chromatin preparation as described earlier [7 (link)]. Nuclei were re-suspended in ChIP-incubation buffer at a concentration of 20 × 10⁶ cells/mL and sheared (seven cycles with each cycle containing 10 s power on and 10 s interval) using Bioruptor®Plus (B01020001, Diagenode, Liege, Belgium). Sonicated chromatin equivalent of 4 × 10⁶ cells was incubated with relevant antibody overnight at 4 °C. Antibodies against P300 (sc-585x, Santa Cruz Biotechnology, Inc., Dallas, Texas, United States), POLII (MMS-126R-500, Covance, Inc., Princeton, New Jersey, United States), H3K27ac (C15410196, Diagenode), H3K4me1 (C15410194, Diagenode), and H3K4me3 (C15410003, Diagenode) were used. ChIP-seq sample preparation and sequencing was performed according to manufacturer’s instructions (Illumina, San Diego, California, United States) and essentially as described [6 (link), 9 (link), 51 (link)] (http://www.blueprint-epigenome.eu).