GSM5627781: GSC23 Input; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde at room temperature for 10 minutes, and the reaction was subsequently quenched with 0.125 M glycine. Then, the cells were washed with cold PBS and suspended with lysis buffer supplemented with Protease Inhibitor Cocktail II. Chromatin was sonicated to obtain DNA fragments with an average length of 200-500 bp by a Bioruptor Pico sonication device (Diagenode) followed by centrifugation for clearing. The soluble fraction of sheared chromatin was diluted 10-fold with ChIP dilution buffer and incubated with 5L H3K4me3 antibody (Cat# 39159, Active Motif) conjugated to EZ-Magna ChIP A/G magnetic beads overnight at 4°C with rotation. Chromatin-captured beads were washed once with low salt wash buffer, once with high salt wash buffer, once with LiCl wash buffer and once with TE buffer for 10 min each by rotation at 4°C. Beads were resuspended in elution buffer and the supernatant was removed by magnet. Proteinase K was added to the eluted DNA followed by incubation at 62°C for 4 hours and 95°C for 10 minutes with shaking. Reverse-crosslinked DNA was purified by using purification columns and quantified by using PicoGreen 628 (Invitrogen). Eluted DNA was used for sequencing library preparation or quantitative PCR (qPCR). Sequencing libraries were prepared using ThruPLEX® NGS Library Preparation Kits (Rubicon Genomics, R400427) according to manufacturer's instruction. Following library amplification, DNA fragments were agarose gel (1.0%) size selected (<1 kb), analyzed using a Bioanalyzer (Agilent Technologies). Libraries were sequenced at Genewiz by using Illumina HiSeq 2500 2x150-bp paired-end reads.