GSM7898068: WT_NPC_Hi-C_Rep2; Mus musculus; Hi-C
3 million cells were lysed in NP40 lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM EDTA, 10 mM Tris pH8, 0.1% NP40) , fixed in 1% formaldehyde (Sigma 252549) at RT for 5 minutes and quenched with 125 mM glycine at RT for 10 minutes. Nuclei were incubated in 0.5% SDS at 62 ℃ for 10 minutes, and quenched with 1.25% Triton X-100 (Sigma 93443) at 37 ℃ for 15 minutes. Chromatin was digested with 100 U of MboI restriction enzyme (NEB R0147) in 1X NEBuffer2 (NEB B7002S) at 37 ℃ overnight with rotation. Enzyme digestion was inactivated with incubation at 65 ℃ for 20 minutes, and nuclei were collected with centrifugation. Restriction fragment overhangs were filled and DNA ends were marked with biotin using End-it DNA Repair Kit (Epicentre ER0720) supplemented with 0.256 mM biotin-14-dATP (Life Technologies 19524-016), 0.4 mM dCTP, 0.4 mM dGTP and 0.4 mM dTTP (NEB N0446S). For proximity ligation, DNA fragments were ligated with 2000 U of T4 DNA ligase (NEB M0202) in 1200 μl of ligation master mix (T4 DNA ligase buffer (NEB B0202), 0.83% Triton X-100, 0.1 mg/ml bovine serum albumin (NEB B9200S)) at RT for 4 hours with slow rotation. After ligase inactivation at 65 ℃ for 5 minutes, nuclei were collected with centrifugation, and the chromatin was reverse-crosslinked in PK lysis buffer (1 M Tris pH8, 50 mM EDTA, 2% SDS, 2 M NaCl) at 65 ℃ for 4 hours, followed by RNase A (Sigma R6513) and proteinase K (Sigma P2308) digestion. DNA was further purified by phenol/chloroform (1:1) and ethanol precipitation, dissolved in buffer EB (Qiagen 19086) and sonicated to ~ 300 base pairs by Covaris E220 ultrasonicator system. For biotin pull-down, 100 μl per sample of 10 mg/ml Dynabeads MyOne Streptavidin T1 beads (Life technologies 65602) was washed with 400 μl per sample of 1 X TWB (5 mM Tris-HCl pH7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), resuspended in 100 μl per sample of 2X binding buffer (10 mM Tris-HCl pH7.5, 1 mM EDTA, 2 M NaCl) and incubated with samples at RT for 15 minutes with rotation. The beads were separated on magnetic stand, washed three times with 1 X TWB (pre-warmed to 55 ℃) and once in 100 μl buffer EB (Qiagen 19086). The beads were collected for DNA end repair with End-it DNA Repair Kit, A-tailing with Klenow Exo-minus (Epicentre KL06041K), adapter (Vazyme N802-01) ligation with Fast-Link kit (Epicentre LK11025) and PCR amplification with VAHTS HiFi Universal Amplification Mix for Illumina (Vazyme N618-02). Supernatant was taken as the library, which was further size-selected with SPRIselect beads (Beckman B23318) and sent for deep sequencing on Illumina NovaSeq 6000 (paired-end, 150 bp).