Instrument: NextSeq 500
Strategy: Hi-C
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: 2x10^8 cells were harvested and fixed with 1% formaldehyde for 20 minutes at room temperature. The reaction was stopped by addition of glycing and incubation for 5 minutes on ice. Cells were snap frozen in liquid nitrogen. Nuclei were permeabilized using with digitonin and SDS and the chromatin was digested with MboI. Restriction fragments were biotinylated whilst being end-repaired and then re-ligated. Crosslinks were reversed and the DNA was fragmented.Biotinylated chimeric DNA fragments were enriched by addition of strepavidin coated magnetic beads and separation with a magnet. Biotinylated DNA was end-repaired using T4 polynucleotide kinase (NEB), T4 DNA polymerase (NEB) and DNA plymerase I, large Klenow fragment (NEB). DNA was reclaimed with a magnet, washed and subsequently polyadenylated at the 3` end using Klenow fragment (3´ to 5´ exo minus) and dATP. DNA fragments bound to the beads were reclaimed with a magnet, washed and annealed TruSeq adapters were ligated to the DNA fragments using Quick Ligase (NEB) and was purified by reclaiming with a magnet. The DNA was eluted from the beads by heating at 98°C for 10 minutes. The library was amplified using Illumina primers and 10 or 15 PCR cycles. The libray was purified using Agencourt AMPure XP beads (Beckman Coulter). Libaries were quantified using Qubit and qPCR and were sequenced on an Illumina flow cell in an Illumina NextSeq aparatus following the manufacturer's instructions.