Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells (1x107) were grown in SUM Medium. The media were then removed and replaced with media containing 1% formaldehyde (EM grade; tebu-bio) and crosslinked for 8 min. Crosslinking was quenched by adding glycine to a final concentration of 0.2 M. The cells were washed with ice-cold PBS, harvested in PBS, and the cell pellet was washed with PBS. The nuclear fraction was extracted by first resuspending the pellet in 10 ml of LB1 buffer (50mM HEPES-KOH [pH 7.5], 140mMNaCl, 1mMEDTA, 10% glycerol, 0.5% NP-40 or Igepal CA-630, and 0.25% Triton X-100) for 10 min at 4C. Cells were pelleted, resuspended in 10 ml of LB2 buffer (10 mM Tris-HCL [pH 8.0], 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), and mixed for 5 min. Cells were pelleted and resuspended in 1ml LB3 buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 0.1%SDS, 1%Triton X-100, 0.1% Na-deoxycholate, 1Mm DTT, 0.25% N-lauroylsarcosine, protease inhibitors and phosphatase inhibitors) and sonicated in a covaris sonicator for 10 min. A total of 30 l of 5M Nacl was added, and lysate was centrifuged for 10 min at 20,000 rcf to purify the debris. The supernatant was then incubated with 50 l of magnetic beads (Life Technologies) prebound with 20 g BRD4 antibody (Bethyl, A301-985A), and immunoprecipitation (IP) was conducted overnight in the cold room. The beads were washed ten times in 1 ml of RIPA buffer and twice in 100mM ammonium hydrogen carbonate (AMBIC) solution. DNA was eluted in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS). Cross-links were reversed overnight at 65C. RNA and protein were digested with 0.2 mg/mL RNase A for 2 hr followed by 0.2 mg/mL Proteinase K for 1 hr. DNA was purified with phenol chloroform extraction and ethanol precipitation. ChIP-seq libraries were prepared using the Rubicon ThruPLEX DNA-seq Kit from 1 ng of purified ChIP DNA or input DNA according to the manufacturer's protocol.