Illumina HiSeq 4000 sequencing; ChIP seq for p63, H3K27ac and BRD4 in L3.6pl, BxPC-3 pancreatic cancer cell lines and ChIP-seq of H3K27ac and BRD4 in Panc-1 pancreatic cell line
ChIP seq for p63, H3K27ac and BRD4 in L3.6pl, BxPC-3 pancreatic cancer cell lines and ChIP-seq of H3K27ac and BRD4 in Panc-1 pancreatic cell line
Briefly, cells were crosslinked with 1% formaldehyde for 20 minutes and quenched by Glycine (125mM final concentration). Upon cell scraping, nuclear pellets were prepared in and washed with the nuclear preparation buffer (150mM NaCl, 20 mM EDTA, 50 mM Tris-HCl (pH 7.5), 0.5% v/v NP-40, 1% v/v Triton-X-100, 20 mM NaF). Then samples were sonicated in sonication buffer (150mM NaCl, 20 mM EDTA, 50 mM Tris-HCl (pH 8), 1% v/v NP-40, 0.5% v/v Sodium deoxycholate, 20 mM NaF, 0.1% SDS) for 30 cycles (in L3.6pl) and 25 cycles (in BxPC-3 and Panc-1) using a Bioruptor Pico (Diagenode) and a cycle setting of 30s on and 30s off. Consequently, samples were precleared by incubation with 50% slurry of Sepharose 4B (GE Healthcare) and incubated with antibody overnight. Antibodies included 1µg of p63 (4A4) (sc-8431, Santa-Cruz), 1µg of H3K27ac (196-050, Diagenode), 2µg of BRD4 (C15410337, Diagenode), and 1µg of Rabbit IgG (C15410206, Diagenode). Sepharose beads with Protein A (for rabbit antibodies) or Protein G (for mouse antibodies, 4A4 p63) were added to samples and incubated for 2 hours followed by washes, de-crosslinking, and DNA extraction.  Extraction of DNA was performed using Roti(R)-phenol/chloroform/Isoamylalcohol (Roth) extraction followed by precipitation in 100% Ethanol and washes with 70% Ethanol.  Libraries for DNA from ChIP were made using the Microplex Library Preparation kit v2 (Diagenode) according to the manufacturer's instructions.
Experimental Factor: immunoprecipitate
anti-BRD4
Experimental Factor: cell line
Panc1