Anti brd4

Chromatin immunoprecipitation was performed as described previously (28 ) with the following modifications. Chromatin was sheared in diluted lysis buffer to 200-500bp using a Covaris M220 Focused-Ultrasonicator with the following parameters: 10 minutes, peak incident power 75, duty factor 10%, 200 cycles/burst. Antibodies for ChIP were obtained from commercially available sources: anti-H3K27Ac (Active Motif, #39133), anti-BRD4 (Active Motif, #39909), anti-c-Jun (Cell Signaling, #9165T), anti-c-Fos (Santa Cruz Biotechnology, #sc-166940), anti-BRD3 (Santa Cruz Biotechnology, #sc-515729), and anti-BRD2 (Cell Signaling, #5848S). 5% of the chromatin was not exposed to antibody and was used as control (input). For ChIP-qPCR analysis DNA quantity for each ChIP sample was normalized against input DNA. For ChIP-seq samples, after DNA purification ChIP-seq DNA libraries were prepared with either the TruSeq ChiP Library Prep Kit (Illumina, #IP-202-1012) or the Accel-NGS 2S Plus DNA Library kit (Swift Bioscience, #21024) and sequenced using 75 bp single-end sequencing on an Illumina Hi-seq 4000.

Protein samples were collected in SDS sample buffer, separated using gel electrophoresis and transferred via wet transfer onto a PVDF membrane. The membrane was blocked with 5% milk in TBST and probed with primary antibodies at 1:1000 dilution overnight at 4°C and secondary HRP antibodies at 1:2000 for 1 hour at RT. Signal was assessed via chemiluminescence with the SuperSignal West Pico PLUS substrate (ThermoFisher, #34580) and visualized on a ChemiDoc MP system (Bio-Rad). Anti-Cas9 antibody (Cell Signaling, #14697), anti-β-actin (Sigma, #A3854), anti-c-Jun (Cell Signaling, #9165S), anti-c-Fos (Santa Cruz Biotechnology, #sc-52), anti-HA-HRP (Santa Cruz Biotechnology, #sc-805), anti-BRD4 (Active Motif, #39909), anti-BRD3 (Santa Cruz Biotechnology, #sc-515729), anti-BRD2 (Cell Signaling, #5848S), and c-Myc (Cell Signaling, #9402) and anti-EGFR (BD Biosciences, #610017) were used for analysis.