GSM8607382: PBMC, low, 243, H3K27ac; Homo sapiens; ChIP-Seq
PBMCs were resuspended in RPMI culture medium and fixed using formaldehyde (1% final concentration, Sigma Aldrich) for 10 minutes at room temperature. Unreacted formaldehyde was quenched with 125 mM glycine and incubated for 5 minutes at room temperature. Cells were washed twice in PBS containing protease inhibitor cocktail (Roche) and 1 mM PMSF (Roche), and subsequently snap frozen in liquid nitrogen. Cell pellets were stored at -80°C for further use. Cells were sonicated at a concentration of 15 million cells/mL using a Bioruptor pico sonicator (Diagenode; 10-20 cycles, 30s on, 30s off), at 4°C. Immunoprecipitation was performed using the MagnaChIP kit (Merck Millipore) according to manufacturer's instruction. In short, 500,000 cells were incubated overnight with 1 mg H3K4me3 (Diagenode) and protein A magnetic beads at 4°C. Beads and chromatin/antibody mixture were washed four times for 5 minutes at 4°C. After washing chromatin was eluted and proteins were degraded using proteinase K. DNA was purified using spin columns and eluted in millliQ. ChIP-seq libraries were prepared using the Kapa Hyper Prep Kit according to manufacturer's protocol, with the following modifications. 2.5 mL of the NEXTflex adaptor stock (600 nM, Bio Scientific) was used for adaptor ligation of each sample. Libraries were amplified with 12-15 PCR cycles followed by a double post-amplification clean-up used to ensure proper removal of adapters. Samples were analyzed for purity using a High Sensitivity DNA Chip on a Bioanalyzer 2100 system (Agilent). Libraries were paired-end sequenced to a read length of 50 bp on an Illumina NextSeq500.