title: GSM6585401: L36_HiCHIP_Rep1; Homo sapiens; OTHER
library_construction_protocol: After crosslinking the cells with 1% formaldehyde, cells were washed and lysed. The digestion was carried out with 200U of MboI, DpnII and HinfI (NEB) at 37°C for 2 hours. The overhangs were filled by adding dCTP, dGTP, dTTP and biotin labeled dATP (Jena biosciences) and DNA polymerase I Large (Klenow) Fragment (NEB). T4 DNA Ligase (NEB) mediated proximity ligation was carried out after the biotin incorporation. The samples were then resuspended in lysis buffer supplemented with protease inhibitors and sonicated. After verifying the size distribution of DNA fragments, the chromatin was precleared with 50% sepharose 4B (GE Healthcare) slurry and incubated with 6 μg of H3K27ac antibody (#196-050, Diagenode). Protein A-sepharose (GE Healthcare) beads were used to capture the immunocomplexes. After several rounds of washes, DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1). Size selection using KAPApure beads (Roche) was performed according to manufacturer's guidelines to exclude big DNA fragments prior to library preparation. Libraries were prepared using KAPA HyperPrep (Roche) kit.  Before library amplification, biotin-labeled library DNA molecules were captured with Streptavidin T-1 beads (Invitrogen). The fragment distribution of HiChIP libraries was determined using Bioanalyzer (Agilent).