GSM9085290: hic_csr1; Neurospora crassa; Hi-C
In situ Hi-C was conducted as described in Tanizawa 2017 with some modifications.  The fixed cells were disrupted with a Mini-Beadbeater-16 (BioSpec Products) and glass beads (Sigma, G8772) in the presence of lysis buffer [50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1x Complete (Roche, 11836170001), and 1 mM PMSF].  The cells were washed twice with 1x PBS and once with 1.11x NEBuffer 3.1 (New England Biolabs).  Nuclei were permeabilized in 1.11x NEBuffer 3.1 containing 0.1% sodium dodecyl sulfate (SDS) at 62°C for 10 minutes.  The SDS reaction was quenched with 1% Triton X-100 at 37°C for 15 minutes.  Genomic DNA within the permeabilized nuclei was digested with 0.1 U/µL DpnII (New England Biolabs, R0543) at 37°C overnight.  The restriction enzyme was heat-inactivated at 62°C for 20 minutes.  DNA fragment cohesive ends were blunted with 0.13 U/µL Klenow fragment (New England Biolabs, M0210) at 37°C for 45 minutes in the presence of 50 µM biotin-14-dATP (Jena Bioscience, NU-835-BIO14) to label DNA fragment ends with biotin.  The resulting blunt-ended DNA fragments were subjected to proximity ligation using 4.17 U/µL T4 DNA ligase (New England Biolabs, M0202) at room temperature for 4 hours.  DNA was treated by 0.5 mg/mL proteinase K (Invitrogen, 25530049) in the presence of 0.5% SDS at 65°C for 4 hours, followed by phenol:chloroform:isoamyl alcohol (Sigma, P2069) extraction and ethanol precipitation. The purified DNA was fragmented by sonication with a Bioruptor UCD-200 (Diagenode) and subsequently purified with a 1:1 volume ratio of AMPure XP (Beckman Coulter, A63881) magnetic beads.  Biotin-labeled DNA fragments were captured using Dynabeads MyOne Streptavidin T1 magnetic beads (Invitrogen, 65601) at 25°C for 15 minutes.  While immobilized on the beads, DNA end-repair was performed with 0.5 U/µL T4 polynucleotide kinase (New England Biolabs, M0201), 0.12 U/µL T4 DNA polymerase (New England Biolabs, M0203), and 0.05 U/µL Klenow fragment (New England Biolabs, M0210) at 25°C for 30 minutes.  This step was followed by dA tailing with 0.5 mM dATP and 0.25 U/µL 3'→5' exo- Klenow fragment (New England Biolabs, M0212) at 37°C for 30 minutes.  The resultant dA-tailed DNA was ligated to NEBNext Adaptor for Illumina (New England Biolabs) using the NEBNext Ultra II Ligation Module (New England Biolabs, E7595) in combination with USER Enzyme (New England Biolabs).  Next-generation sequencing libraries were amplified with four cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544) and NEBNext Multiplex Oligos for Illumina (New England Biolabs).  Post-amplification, libraries were purified with a 0.9:1 volume ratio of AMPure XP beads (Beckman Coulter, A63881).  Where applicable, libraries were multiplexed prior to sequencing on an Illumina NextSeq 500 platform.