title: GSM8542522: K562, Replicate2, H3K27ac HiChIP; Homo sapiens; OTHER
library_construction_protocol: For H3K27ac HiChIP libraries, we grew NIH3T3 (mouse) cells and human cell lines A375, A549, HT29, K562, MDA-MB-231 and PC-9 in the appropriate media.  We fixed cells in 1% formaldehyde for 10min and quenched to a final concentration of 125nM glycine. We mixed 2 million fixed mouse cells with 10 million of each fixed human cancer cell line. The cells were lysed in 0.5% SDS, quenched with 10% Triton X-100, and digested with MboI (NEB R0147M). The DNA overhangs were blunted, biotinylated (ThermoFisher 19524016), and ligated. Nuclei were spun down, resuspended in nuclear lysis buffer and sonicated using a Covaris LE220 with the following conditions: Fill level 10, PIP 450, Duty factor 30, CPB 200. We incubated the sheared DNA with Dynabeads Protein A (ThermoFisher 10001D) for 2 hours at 4 °C. We then placed the tubes on a magnet and the supernatant was kept. We performed immunoprecipitation with a cross-species reactive H3K27ac antibody (Active Motif 39133). The samples were incubated with the antibody overnight at 4 °C. We then washed, eluted and treated the samples and treated with Proteinase K. We purified the samples using Zymo DNA Clean & Concentrator Biotin capture was performed with Dynabeads M-280 Streptavidin (ThermoFisher 11205D), followed by library preparation as previously described (Gryder, Khan and Stanton, 2020). We purified the amplified libraries with Illumina Sample Purification Beads. We sequenced the libraries using paired-end reads with NovaSeq 6000 or NextSeq 500.