GSM9361303: WT_control; Schizosaccharomyces pombe; Hi-C
Fission yeast cells (approximately 5 x 108 cells at OD595 = 0.5) were fixed with 3% pFA at 26°C for 10 minutes and disrupted with glass beads using Mini-Beadbeater-16 (BioSpec Products) in lysis buffer [50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1 mM PMSF, and EDTA-free Protease Inhibitor Cocktail (Roche)].  After washing with 1 ml PBS solution, the cells were further fixed with 3 mM disuccinimidyl glutarate (Thermo Fisher Scientific) in 2.5 ml PBS solution at 30°C for 40 minutes.  The fixed cells were incubated at 62°C for 7 minutes with 0.1% SDS and subsequently with 1% Triton-X 100.  The permeabilized cells were incubated with 25 units of MboI, HinfI, and MluCI at 37°C overnight.  The pellet containing restriction enzyme-digested DNA fragments was treated with 150 μM biotin-14-dATP, dCTP, dGTP, and dTTP and incubated with 20 units of Klenow fragment (NEB) at 37°C for 45 minutes, followed by proximity ligation using 2,500 units of T4 DNA ligase (NEB) at room temperature for 4 hours with a gentle rotation.  After ligation, the pellet was subjected to proteinase K treatment at 68°C overnight in the presence of 0.5 M NaCl. DNA was purified by phenol/chloroform extraction and ethanol precipitation.  The purified DNA was sheared into 100–500 bp fragments by Bioruptor UCD-200 (Diagenode).  Biotin-labeled DNA was purified using Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific).  Sequencing adapters were ligated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) while DNA was bound to the beads.  Sequencing libraries were amplified by PCR using the NEBNext Ultra II Q5 Master Mix (NEB) with NEBNext Multiplex Oligos for Illumina (NEB).