GSM7911748: WAPLKO_C; Homo sapiens; OTHER
10 million cells were collected and crosslinked with freshly made 1% formaldehyde, and crosslinking was quenched with 125 mM glycine. Crosslinked cells were lysed with iced-cold Hi-C lysis buffer at 4°C for 30 minutes with rotation. Pelleted nuclei were harvested and digested with 375U of MboI restriction enzyme (NEB, R0147) at 37°C for 2 hours with rotation. Next, DNA ends were filled in and marked with biotin-dATP (Thermo, 19524016). T4 DNA ligase (NEB, M0202) was used for proximity ligation at room temperature for 4 hours with rotation. After ligation, pelleted nuclei were lysed with iced-cold nuclear lysis buffer and sonicated with Covaris M220 for 240 seconds. ChIP dilution buffer was used to dilute each sample to achieve a SDS concentration of 0.1%. After preclearing with Protein A magnetic beads, 7.5 µg of anti-H3K27ac antibody (abcam, ab4729) were added for every 10 million cells and incubated overnight at 4°C. Protein A magnetic beads were used to capture DNA-protein complexes. Beads were washed (three times each in Low Salt Wash Buffer, High Salt Wash Buffer, and LiCl Wash Buffer) and DNA was eluted in Elution Buffer. Crosslinks were then reversed via Proteinase K digestion, and DNA was purified with the QIAquick PCR purification kit (Qiagen). Samples were prepared for sequencing using the  Illumina Tagment DNA  Enzyme and Buffer Kits (Illumina, 20034197) per the manufacturer's instructions.