Pore-C sequencing of Dalbergia oliveri (Oxon_Daoli_PoreC_3)
Pore-C library was prepared with the protocol and reagents described by Belaghzal et al. with minor modifications. We harvested 2 g of fresh leaf from the same reference individual as for the Nanopore library and immediately cross-linked the finely chopped tissues in 1% formaldehyde for 20 minutes. The cross-linking was then quenched with 125 mM glycine for 20 minutes and the samples were then ground in liquid nitrogen with a mortar and a pestle. Cell nuclei were isolated with a buffer containing 10 mM Trizma, 80 mM KCl, 10 mM EDTA, 1 mM spermidine trihydrochloride, 1 mM spermine tetrahydrochloride, 500 mM sucrose, 1% (w/v) PVP-40, 0.5% (v/v) Triton X-100, and 0.25% (v/v) -mercaptoethanol, and then passed through a 40 m cell strainer. The suspension was centrifuged at 3,000 g, according to the estimated genome size of ~700 Mbp. Chromatin was denatured with the restriction enzyme NlaIII at a final concentration of 1 U/L (New England Biolabs, United Kingdom) at 37C for 18 hours. The enzyme was heat-denatured at 65C for 20 minutes at 300 rpm rotation. Proximity ligation, protein degradation, decrosslinking, and DNA extraction were performed according to the original Belaghzal protocol. The Pore-C library was prepared with the Oxford Nanopore Technologies Ligation Sequencing Kit (SQK-LSK110), then sequenced on R10.3 (FLO-109D) Nanopore flow cells on a GridION sequencer.