title: HiC library for sample HG03486  (Mende)
design: Five million human lymphoblastoid  cells derived from an individual of Mende descent (Coriell code HG03486) were  resuspended in cold PBS before crosslinking with one-percent paraformaldehyde  (EMS Catalog 15714). Crosslinked cells were lysed in a hypertonic buffer with  one-percent SDS before binding crosslinked chromatin to SPRI beads in 18%  PEG-8000. Bead-bound chromatin was thoroughly washed and then digested with  DpnII restriction enzyme (20 U, NEB Catalog R0543S). Biotin-11-dCTP (ChemCyte  Catalog CC-6002-1) was incorporated by DNA Polymerase I, Klenow Fragment (10  U, NEB Catalog M0210L) and intra-aggregate ligation was achieved overnight by  T4 DNA Ligase (4000 U, NEB Catalog M0202T). Proximity-ligated DNA was  isolated by SPRI bead purification after crosslink reversal with Proteinase K  (Qiagen Catalog 19133) in an eight percent SDS solution. The purified  proximity-ligation products were randomly reduced to Illumina library insert  size via Diagenode Bioruptor NGS sonication platform. TruSeq library preparation  was accomplished using the NEBNext Ultra II DNA Library Prep Kit for Illumina  (NEB Catalog E7645S) and a custom Y-adaptor. Adaptor ligation products were  SPRI bead purified before capturing biotinylated molecules using Dynabeads  MyOne Streptavidin C1 beads (ThermoFisher Catalog 65002). After enrichment of  biotinylated products by buffer exchange, the streptavidin-bound DNA was used  as input to indexing PCR with KAPA HiFi HotStart ReadyMix (Catalog no.  KK2602) and TruSeq indexing adaptors with unique index sequences. The  resulting library molecules were purified and simultaneously size-selected  using SPRI beads before sequencing.