GSM6067340: PBRM1_786O_PBRM1WT_ChIPSeq_Rep2; Homo sapiens; ChIP-Seq
For each protein of interest, approximately 2x107 cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and stopped by adding glycine to a final concentration of 0.2M. Chromatin was extracted and sonicated to ~500bp (Vibra cell, SONICS). The total volume of immunoprecipitation was 2 ml and the amount of antibody used was 20 µg. The input DNA was precleared with protein G Dynabeads (LifeTechnologies) for 2 hours at 4°C and then incubated with antibodies overnight at 4°C. Protein G beads were added the following day and mixture was nutated for 3 hours at 4°C. The beads were washed 6 times with wash buffer at room temperature. At least 10 ng of the amplified DNA was used with NEBNext ChIP-Seq library prep reagent set (NEB). Each library was sequenced to an average depth of 30-50 million reads on HiSeq2500 or HiSeq4000.