HiChIP Profiling of H3K27Ac and RelA

HiChIP was performed as previously described^{53 (link)}, using antibodies against 1 μg of H3K27ac (Abcam) or 5 μg of RelA (Cell Signaling Technology). Briefly, cells (2 × 10⁶ for H3K27ac, 1 × 10⁷ for RelA) were crosslinked with 1% formaldehyde (Sigma) for 10 min and subsequently quenched with 0.125 M glycine (Invitrogen). Chromatin was digested using MboI restriction enzyme (NEB), followed by biotin incorporation with Biotin-14-dATP (Jena bioscience) in end-repair step, ligation, and sonication. Sheared chromatin was then incubated with antibodies recognizing H3K27ac or RelA at 4 °C overnight. Chromatin-antibody complexes were captured by Protein A and G magnetic bead (Invitrogen) and subsequently washed with Low Salt Wash Buffer, High Salt Wash Buffer, and LiCl Wash Buffers before being eluted. DNA was purified with the MinElute PCR Purification Kit (Qiagen) and quantified using the Qubit dsDNA HS Assay Kit (Invitrogen). Subsequently, 50–150 ng was used for capture with Dynabeads MyOne Streptavidin C-1 (Invitrogen) and an appropriate amount of Tn5 enzyme (Illumina) was added to captured DNA to generate sequencing library. Each library was paired-end sequenced (100 bp) on Illumina NovaSeq6000 platform. Two biological replicates were performed for each condition.