PoreC proximity ligation sequencing of D. pachys nuclear DNA
A sequencing library for D. pachys Pore-C was generated by extracting DNA crosslinked to chromatin that was subsequently digested by a restriction enzyme and religated. Briefly, D. pachys animals suspended in M9 buffer (22.0 mM KH2PO4, 42.3 mM Na2HPO4, 85.6 mM NaCl, 1 mM MgSO4) were flash-frozen in liquid nitrogen and ground up using mortar and pestle to release cellular contents and nuclei. Chromatin and cellular components were crosslinked using 1% w/v formaldehyde in PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) at room temperature for 10 minutes. After quenching the formaldehyde with 1% w/v glycine and the removal of formaldehyde, nuclei were permeablized using 0.2% v/v IGEPAL (with 10 mM Tris-HCl and 10 mM NaCl, pH 8) supplemented with protease inhibitor cocktail P8340 (Sigma, St. Louis, MO) and heat-treated at 65 C for 1 hr. Chromatin was denatured using 0.1% w/v sodium dodecyl sulfate (SDS) and the DNA was then digested by NlaIII (New England Biolabs, Ipswich, MA) at 37C overnight. NlaIII was subsequently deactivated by heat (65C for 20 minutes) and NlaIII-digested ends were allowed to anneal by slowly cooling to room temperature before ligation using T4 DNA ligase (New England Biolabs, Ipswich, US). Finally, proteins were digested from the crosslinking ligation reaction by incubating with 1 g/L proteinase K (Invitrogen, Waltham, MA) at 56C for 18 hours. DNA from this preparation was purified using standard phenol-chloroform extraction and resolubilized with TE buffer. The purified, proximity-ligated DNA was ligated to Nanopore adapters per the manufacturers instructions. The library was then sequenced on an Oxford Nanopore R9.4.1 flowcell (FLO-MIN106) and basecalled using Guppy (v 6.0.7).