title: GSM2612322: ChIP-Seq_786-M2B_H3K27ac; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were crosslinked in 1% formaldehyde for 10min at room temperature, followed by 5 min quenching with 0.125M glycine. Cells were washed twice with PBS, the supernatant was aspirated and cell pellets were frozen in liquid nitrogen and stored at -80°C. 100µl of Protein A/G magnetic beads (Thermo 26162) were blocked with 0.5% BSA in PBS, followed by incubation with the antibody. Cross-linked cells were resuspendend and sonicated in lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, and 1% Triton X-100). Sonication was performed using Bioruptor (Diagenode) for 14 cycles (30’’ on/ 30’’ off) at max output to obtain fragments of 100-500bp. Sonicated lysates were cleared and incubated overnight at 4°C with antibody-bound magnetic beads. Beads were sequentially washed three times with low salt buffer (50mM HEPES pH7.5, 140mM NaCl, 1% Triton) and once with high salt buffer (50mM HEPES pH7.5, 500mM NaCl, 1% Triton). DNA was eluted in elution buffer (50mM NaHCO3, 1%SDS) and cross-links were reversed for 3h (65°C, 1000rpm shaking). DNA was purified using the QuickClean II PCR Extraction Kit (Genescript L00419-100) according to the manufacturer’s recommendations and eluted with 100µl H2O. Purified ChIP DNA was used to prepare Illumina multiplexed sequencing libraries with the KAPA Hyper Prep Kit (KR0961) Illumina platforms sample preparation protocol (v1.14). Briefly, DNA was end-repaired and A-tailed to produce end-repaired, 5’-phosphorylated, 3’-dA-tailed, dsDNA fragments. These 3’-dA-tailed library fragments were then ligated with dsDNA adapters with 3’dTMP overhangs. After adapter ligation, libraries were size selected to 150-350bp using AMPure XP reagent (Agencourt A63880) according to the protocol recommendations. Size selected libraries were amplified for 15 cycles using the KAPA HiFi HotStart ReadyMix. PCR libraries were pooled in equimolar concentrations and sequenced.