GSM8402320: BxPC3,plusIFNγ, Replicate4, H3K27ac HiChIP; Homo sapiens; OTHER
We prepared HiChIP cell libraries using H3K27ac AQuA-HiChIP method (Gryder, Khan and Stanton, 2020). We cultured BxPC-3 cells in RPMI media supplemented with 10% Serum Plus-II (Thermo Fisher) only or in RPMI media supplemented with 10% Serum Plus-II (Thermo Fisher) and 100 IU/mL IFNγ (BioLegend) for 2 days. For absolute quantification (AQuA-HiChIP), we grew 3T3 mouse fibroblasts in DMEM media (supplemented with 10% Serum Plus-II (Thermo Fisher Scientific) with no IFNγ. Then, we fixed cells in 1% formaldehyde (Sigma-Aldrich) for 10 mins and quenched to a final concentration of 125nM glycine (Sigma-Aldrich). We spiked in 2 million fixed mouse cells (with no IFNγ treatment) with 10 million of fixed treated-BxPC-3 cells or non-treated cells. The cells were lysed in 0.5% SDS (Sigma-Aldrich), quenched with 10% Triton X-100 (Sigma-Aldrich), and digested with MboI (NEB R0147M). Next, 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 program: 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 to preclear the samples. 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 and Dynabeads Protein A overnight at 4 °C. We then washed, eluted and treated the samples with Proteinase K (New England Biolabs). 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 S1 2x100 kit to generate 100-200 million read pairs per sample. H3K27ac HiChIP