H3K27ac HiChIP of homo sapiens: HAP1
we fixed ten million HAP1 cells in 1% formaldehyde for 10 minutes and quenched to a final concentration of 125 nM glycine. The cells were lysed in 0.5% SDS, quenched with 10% Triton X-100, and digested with 200 units MboI (NEB R0147M) at 37C for 2 hours to produce blunt ends. After heat inactivation at 62C for 20 minutes, the blunt DNA overhangs were biotinylated using DNA Polymerase I Large (Klenow) Fragment (NEB M0210L), 288 M biotin-dATP (Thermo 19524016), dTTP, dGTP, and dCTP at 37C for 1 hour. Biotinylated products were ligated using T4 DNA ligase (NEB B0202S) at room temperature for 4 hours. Nuclei were spun down, resuspended in nuclear lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS) 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 (Thermo 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. Dynabeads Protein A (Thermo 10001D) were added to the samples and incubated for 2 hours at 4 C to bind the DNA with H3K27ac antibodies. The beads with bound DNA were washed, the DNA was eluted, and the DNA was treated with Proteinase K (NEB P8107S). We purified the samples using DNA Clean & Concentrator (Zymo D4014). Biotin capture was performed with Dynabeads M-280 Streptavidin (Thermo 11205D), followed by library preparation. We purified the amplified libraries with Sample Purification Beads (Illumina).