HiC of Ophiodon elongatus
Hi-C library was generated using the Phase Genomics Proximo Animal kit version 4.0. Approximately 500 mg tissue was finely chopped and then crosslinked for 15 minutes at room temperature with end-over-end mixing in 1 mL of Proximo crosslinking solution. Crosslinking reaction was terminated with quenching solution for 20 minutes at room temperature again with end-over-end mixing. Quenched tissue was rinsed once with 1X Chromatin Rinse Buffer (CRB). Tissue was transferred to a liquid nitrogen-cooled mortar and ground to a fine powder. Powder was resuspended in 700 L Proximo Lysis Buffer 1 and incubated for 20 minutes with end-over-end mixing. A low-speed spin was used to clear the large debris and the chromatin containing supernatant transferred to a new tube. Following a second higher speed spin, the supernatant was removed and the pellet containing the nuclear fraction of the lysate was washed with 1X CRB. After removing 1X CRB wash, the pellet was resuspended in 100 L Proximo Lysis Buffer 2 and incubated at 65C for 15 minutes. Chromatin was bound to Recovery Beads for 10 minutes at room temperature, placed on a magnetic stand, and washed with 200 L of 1X CRB. Chromatin bound on beads was resuspended in 150 L of Proximo fragmentation buffer and 2.5 L of Proximo fragmentation enzyme added and incubated for 1 hour at 37C. Reaction was cooled to 12C and incubated with 2.5 L of finishing enzyme for 30 minutes. Following the addition of 6 L of Stop Solution, the beads were washed with 1X CRB and resuspended in 100 L of Proximo Ligation Buffer supplemented with 5 L of Proximity ligation enzyme. Proximity ligation reaction was incubated at room temperature for 4 hours with end-over-end mixing. To this volume, 5 L of Reverse Crosslinks enzyme was added and the reaction incubated at 65C for 1 hour. After reversing crosslinks, the free DNA was purified with Recovery Beads and Hi-C junctions were bound to streptavidin beads and washed to remove unbound DNA. Washed beads were used to prepare paired-end deep sequencing libraries using the Proximo Library preparation reagents. Library fragment size and concentration are determined using TapeStation capillary electrophoresis and quantitative PCR. Prior to production sequencing, library performance is determined by low-pass sequencing, generating ~200,000 read pairs of sequencing data on an Illumina iSeq System. Resulting reads are mapped to a reference assembly and analyzed with Phase Genomics open-source QC tool, hic_qc. Libraries receiving a Sufficient quality judgement from hic_qc are advanced to production sequencing.