CH Campala Lr22a 2D chromosome Chicago library raw reads
250 ng of chromosome 2D HMW DNA (mean fragment length ~100 kb) was reconstituted into chromatin in vitroand fixed with formaldehyde. Fixed chromatin was digested with MboI, the 5 overhangs filled in with biotinylated nucleotides, and then free blunt ends were ligated. After ligation, crosslinks were reversed and the DNA was 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 mean fragment size and a sequencing library was generated using NEBNext Ultra enzymes (New England BioLabs) and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment. The library was then sequenced on an Illumina HiSeq 2500 (rapid run mode) to produce 145 million 150-bp paired-end reads, which provided 30 physical coverage of the chromosome (150 kb pairs).