Hi-C Sequencing Library Preparation

A Chicago (RRID:SCR_014941) library was prepared as described in [34 (link)]. Briefly, ∼500 ng high molecular weight gDNA was reconstituted into chromatin in vitro and fixed with formaldehyde. Fixed chromatin was digested with DpnII, the 5′ overhangs were filled in with biotinylated nucleotides, and then free blunt ends were ligated. After ligation, crosslinks were reversed and the DNA purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ∼350-bp fragments and sequencing libraries were generated using the NEBNext Ultra II kit with Illumina-compatible indices. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina HiSeq X Ten (RRID:SCR_016385) to produce 467 million 2 × 150-bp paired-end reads.