In situ Hi-C Protocol for Chromatin Interaction Analysis

We used the in situ Hi-C protocol as described in Rao et al.^{48 (link)}. In brief, ~3 × 10⁶ RPE cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by an additional 5 min with 200 mM glycine in phosphate-buffered saline (PBS). Fixed cells were permeabilized in Hi-C lysis buffer (10 mM Tris–HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA630; 1× protease inhibitor cocktail [Sigma]) on ice. The cells were treated with 100 U of MboI (New England Biolabs) for chromatin digestion, and the ends of digested fragments were labeled with biotinylated nucleotides followed by ligation. After DNA reverse crosslinking and purification, ligated DNA was sheared to a size of 300–500 bp using a Covaris S2 focused-ultrasonicator (settings: Duty Cycle, 10%; Intensity, 4; Cycles per Burst, 200; Duration, 55 s). The ligated junctions were then pulled down with Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific). The pulled-down DNA was end-repaired, ligated to sequencing adaptors, amplified on beads, and purified using a Nextera Mate Pair Sample Preparation kit (Illumina) and Agencourt AMPure XP (Beckman Coulter). DNA was then sequenced to generate paired-end 150-bp reads using the Illumina HiSeq-2500 or X Ten system.