title: GSM5750370: rec8_F204S_3Flag_0min_PMSF_HiC; Schizosaccharomyces pombe; Hi-C
library_construction_protocol: Asynchronous or meiotic cell culture of 4x10^8 cells was fixed with 3% paraformaldehyde at 26°C for 10 minutes. The fixed cells were disrupted by glass beads (BioSpec Products, Mini-Beadbeater-16) in lysis buffer [50 mM HEPES/KOH pH7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1x Complete (Roche, 11836170001), 1 mM PMSF]. The lysed cells were collected by centrifugation and permeabilized by resuspension with 1.11× NEBuffer 2 (NEB, B7002) containing 0.1% SDS at 62°C for 7 minutes. After SDS was quenched by adding 1/9 volume of 10% Triton X-100, the permeabilized cells were treated with 25 units of MboI (NEB, R0147) at 37°C overnight. The enzyme was inactivated by incubating at 62°C for 20 minutes. After centrifugation, the pellet containing restricted DNA fragments was used in the following procedure. A half volume of fill-in mix [1× NEBuffer 2, 150 µM biotin-14-dATP (Jena Bioscience, NU-835-BIO14), 150 µM dCTP, 150 µM dGTP, 150 µM dTTP, 0.4 units/µl Klenow fragment (NEB, M0210)] was added to the pellet and incubated at 37°C for 45 minutes. Subsequently, 3 volumes of ligation mix [1.33× T4 DNA ligase buffer (NEB, B0202), 1.11% Triton X-100, 133 µg/ml BSA (NEB, B9001), 5.56 units/µl T4 DNA ligase (NEB, M0202)] was added and incubated at room temperature (~25℃) for 4 hours. After the ligation, the pellet was collected by centrifugation, resuspended in Proteinase K solution [10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% SDS, 0.5 mg/ml proteinase K (Invitrogen, 25530049)], and incubated at 65°C overnight. DNA was purified by phenol-chloroform treatment followed by ethanol precipitation, and then fragmented by sonication with Bioruptor (Diagenode, UCD-200). After the biotin-labeled DNA was pulled down with Dynabeads Streptavidin T1 (Invitrogen, 65601), the beads were washed twice with TNB (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20) at 55°C for 2 minutes. To ligate the Illumina sequencing adaptors to the biotinylated DNA fragments, the beads were successively treated with end repair mix [1× T4 DNA ligase buffer, 1 mM dATP, 0.5 mM each dNTP, 0.5 units/µl T4 polynucleotide kinase (NEB, M0201), 0.12 units/µl T4 DNA polymerase (NEB, M0203), 0.05 units/µl Klenow fragment] at 25°C for 30 minutes, dA-tailing mix [1× NEBuffer 2, 0.5 mM dATP, 0.25 units/µl Klenow exo- (NEB, M0212)] at 37°C for 30 minutes, and Illumina adaptor ligation mix [0.075x End repair reaction buffer (NEB, E6050), 0.32× Ligation master mix (NEB, E7595), 0.011× Ligation enhancer, 45 nM NEBNext adaptor for Illumina] at 20°C for 15 minutes followed by 0.032 units/µl USER treatment at 37°C for 15 minutes, with two-time washes with TNB at 55°C for 2 minutes after each reaction. The resultant adaptor-ligated Hi-C library was amplified by PCR with 5-7 cycles with Q5 master mix (NEB, M0544) and index primers (NEB, E7335/E7500/E7710/E7730) before 37-bp paired-end sequencing with NextSeq 500 (Illumina).