HiChIP of human colon normal tissue
HiChIP was performed as described previously (Mumbach et�al., 2016). Briefly, crosslinked samples were lysed in Hi-C lysis buffer and chromatin was permeabilized in 0.5% SDS at 63C for 10�minutes. Chromatin was digested with MboI for 2 hours at 37C. Overhangs were filled in and marked with Biotin-dATP (ThermoFisher 19524016), and ends were ligated with T4 DNA Ligase (NEB M0202) for 4 hours at room temperature. Nuclei were pelleted and lysed and chromatin was sheared on the Covaris E220 with the following conditions: Fill level 5, Duty Cycle 5, PIP 140, Cycles/burst 200, Time 4�minutes. Samples were clarified, diluted in ChIP dilution buffer and precleared with�Protein G�beads (Invitrogen 11205D) for 1 hour at 4C. Samples were cleared on magnet and supernatant was added to antibody. Chromatin was ChIP�d overnight at 4C with rotation. Protein G beads were added and incubated for 2 hours at 4C with rotation. Beads were washed with low salt, high salt and LiCl buffer. Sample was eluted in ChIP elution buffer, treated with Proteinase K and crosslinks were reversed. DNA was purified using the Zymo clean & concentrate kit (DCC-100). Streptavidin M280 beads (Invitrogen 11205D) were washed and resuspended in 2x�biotin�binding buffer and DNA was bound for 15�minutes at room temperature. Beads were washe and Tn5 fragmentation was carried out as per Mumbach et�al.,2016, with dilutions of Tn5 to process low input samples. Libraries were amplified using the Nextera�DNA Library�Prep kit (Illumina). Material was cleaned up using Ampure XP beads. Libraries were sequenced on NextSeq500 (150 cycle kit) or the HiSeq2500 (high output; 200 cycle kit).