Halisarca dujardinii Hi-C, fresh vs frozen tissue: fresh, rep. 2
Cell fixation was carried out in a final volume of 10 mL fixation buffer (artificial seawater containing 1% formaldehyde) at room temperature with gentle rotation. The reaction was quenched by adding 2.5 M glycine to a final concentration of 0.2 M and incubated for 5 minutes at room temperature with rotation. Fixed cells were pelleted twice and resuspended in ice-cold 1 PBS.Cells were then lysed on ice for 25 minutes using 250 L of lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40) supplemented with freshly added protease inhibitors. Following one wash with lysis buffer, cells were pelleted and resuspended in 0.5% SDS, then incubated for 10 minutes at 62C without rotation. Chromatin digestion was performed by adding 100 U of MboI (New England Biolabs) in 250 L of NEB2 buffer per sample, followed by overnight incubation at 37C with constant agitation. The resulting DNA overhangs were filled in and labeled using biotin-14-dATP and DNA Polymerase I, Large (Klenow) Fragment, along with a nucleotide mix. After pelleting the nuclei, a ligation mix (1 NEB T4 DNA ligase buffer, 1% Triton X-100, BSA, and 400 U/L T4 DNA Ligase) was added, and DNA fragments were ligated by overnight incubation at room temperature with gentle rotation. DNA was extracted using the phenol-chloroform method. Samples were sheared using a Covaris LE220 instrument (fill level: 10, duty cycle: 15, PIP: 500, cycles/burst: 200, time: 58 s). Fragments of 300-500 bp were size-selected using AMPure XP beads (Beckman Coulter). Biotinylated fragments were captured using Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific). Library preparation and final amplification were conducted with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturers instructions. A mock PCR was performed to optimize the number of amplification cycles (eight cycles). Prior to sequencing, library size distribution was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). The resulting in situ Hi-C libraries were sequenced using 2 150 bp paired-end reads on the Illumina HiSeq platform