Chicago library preparation and sequencing
Dovetail Genomics LLC prepared a Chicago library as described previously
(Putnam et al., 2016). Briefly, approximately 500 ng of high molecular
weight gDNA (mean fragment length = 80 kb) from an individual female
beetle (NCBI BioSample: SAMN14918906) was reconstituted into chromatinin vitro and fixed with formaldehyde. Fixed chromatin was
digested with DpnII , 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 associated
protein. Purified DNA was treated to remove biotin that was not internal
to ligated fragments. The DNA was then sheared to approximately 350 bp
mean fragment length and sequencing libraries were generated using
NEBNext Ultra enzymes and Illumina-compatible adapters.
Biotin-containing fragments were isolated using streptavidin beads
before PCR enrichment of the library. The library was sequenced on an
Illumina HiSeq X platform to produce 510 million 2x150 bp paired-end
reads (NCBI SRA: SRR11965410), which provided 747 x physical coverage of
the genome (1-100 kb pairs).