GSM9056443: VCaP_H2BK16ac_abcam; Homo sapiens; ChIP-Seq
1-2 million cells were used per ChIP reaction with 2 or 4ug of antibody. Briefly, cells were crosslinked for 10 minutes in a 1% formaldehyde solution, followed by termination with 1/10th the volume of 1.25M glycine for 5 minutes at room temperature. Following this, the cells were lysed and sonicated (Bioruptor Pico, Diagenode) with a desired chromatin size of about 200bp. Sheared chromatin was then incubated with desired antibodies overnight at 4 °C. Next day, the ChIP-DNA was de-crosslinked and purified using the Diagenode iPure Kit V3, following the manufacturer's protocol. Utilizing the manufacturer's instructions (Illumina), purified DNA was prepared for sequencing. About 1-10ng of ChIP DNA were converted to blunt-ended fragments using T4 DNA polymerase, Escherichia coli DNA polymerase I large fragment (Klenow polymerase), and T4 polynucleotide kinase (New England BioLabs (NEB)). Klenow fragment (3′ to 5′ exo minus; NEB) was used to add a single adenine base to fragment ends, followed by ligation of Illumina adaptors (Quick ligase, NEB). PCR enriched the adaptor-ligated DNA fragments using the Illumina Barcode primers and Phusion DNA polymerase (NEB). PCR products were size-selected using 3% NuSieve agarose gels (Lonza) followed by gel extraction using QIAEX II reagents (Qiagen). Quantified libraries quality-checked using the Bioanalyzer 2100 (Agilent) and sequenced on the Illumina HiSeq 2500 Sequencer (125-nucleotide read length).