GSM7698411: SS; Homo sapiens; Hi-C
Crosslink 1 million cells with 37% formaldehyde(sigma, F8775) to a final concentration of 1% and incubate for 10 min at room temperature. Add glycine(Yuanye, S20159) to a final concentration of 0.125M, and terminate the formaldehyde crosslinking reaction at room temperature for 5 min. Cells were pelleted by spinning at 400g for 5 min at 4°C. The pellet was washed with cold PBS. Liquid nitrogen quick-freeze, placed at -80 degrees for reserve.fixed cells were incubated in 500μl of cold lysis buffer (10mM Tris-HCl pH 8.0, 10mM NaCl, 0.2% Igepal CA630 and protease inhibitors (MCE-HY-K0010)) on ice for 25 min. Cells were pelleted by spinning at 400g for 5 min at 4°C. The pellet was washed twice with 1xNEBuffer2. Cells were resuspended in 50μl of 0.5%SDS and incubated for 8 min at 62°C. Next, reactions were terminated with 145μl of water and 25μl of 10%Triton X-100 (Sigma, 93443) at 37°C for 15min. Sample was digested overnight with 25μl of 10X NEBuffer2 and 20μl of MboI(NEB, R0147, 5U/µl) at 37 °C with rotation. Incubate the reactants at 62°C for 20min to inactivate MboI, then cold to room temperature.To complement the bases at the sticky end and label biotin at the DNA end, add 50μl of the mixture（37.5μl 0.4 mM biotin-14-dATP, 1.5μl of 10mMdCTP, 1.5μl of 10mM dGTP, 1.5μlof 10mM dTTP, and 8μl of 5U/μl DNA polymerase I, large (Klenow) fragment(NEB M0210)）and incubating at 37°C for 4h. After that, ligation is performed by adding 120μl of 10XNEB T4 DNA ligase buffer(NEB,B0202L), 100μl of 10% Triton X-100, 637.8μl of water, 1.2μl of 10%BSA(Sigma-Aldrich SRE0098) and 5μl of 400 U/μl T4 DNA ligase(NEB,M0202L) and incubating for 6h at room temperature. De-crosslinking was performed by adding 50μL of 20mg/mL proteinase K (NEB, P8107) and 120μL of 10%SDS incubate at 55°C for 30min. And then 130μL of 5M NaCl was added and incubated at 68 °C overnight. Samples were cooled to room temperature and divided into three equal portions. Then, 800μl of pure ethanol and 50μl of 3M sodium acetate pH5.2 were added to each tube which were subsequently incubated for 30 min at -80°C. Spin the tubes at maximum speed for 15 min at 4°C and wash twice with 70% ethanol. Resuspend the resulting pellet in 130μl of 10mM Tris-HCl, pH8.0 and incubate at 37 °C for 15 min.DNA was sheared using an ME220 Covaris Focused-ultrasonicator to a fragment size of 300–500 bp. Sheared DNA was size selected using VAHTS® DNA Clean Beads N411. Elution was done with hot (65°C) 70μl EB buffer. Next, the sample is transferred to a DNA LoBind tube (Eppendorf, 30108418) for related experimental manipulation. Take 150ul of T1 beads (MyOne Streptavin T1 Beads, Invitrogen, 65601) into a DNA LoBind Tubes and add 500ul 1x TWB buffer (5mM Tris-HCl (pH 7.5); 0.5mM EDTA; 1M NaCl; 0.05% Tween 20) wash twice. Resuspend the T1 beads with 70ul 2× Binding Buffer (10mM Tris-HCl (pH 7.5), 1mM EDTA, 2M NaCl) and combined with 70μl Hi-C DNA from the previous step. The mixture was incubated at room temperature for 15min . After which the magnet adsorbed to remove the supernatant; Wash twice with 200ul 1x TWB buffer and once with 200ul EB buffer. Finally, resuspend the beads with 50ul of water and transfer to a 200ul PCR tube. Library construction using the VAHTS Universal DNA Library Prep Kit (Vazyme ND607) and VAHTS Multiplex Oligos Set 4/5 kits (Vazyme N321/N322) according to manufacturer's instructions