GSM7566845: AID-WT_ZNF143,HiChIP; Homo sapiens; OTHER
Cells were fixed by 1% formaldehyde for 10 minutes at room temperature with slow rotation and quenched by 125 mM glycine. Cells were centrifuged at 800g for 5 minutes and washed once using ice-cold PBS. Cells were suspended using 1 mM ice-cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40, 1x protease inhibitors) for 15 minutes at 4 ℃ with slow rotation and centrifuged at 2,500g, 4 ℃ for 5 minutes to remove supernatant. The above lysis step was repeated again used to fully lyse cells. 100 μL of 0.5% SDS solution was used to resuspend cells and incubation at 62 ℃ for 10 minutes. 240 μL water and 50 μL of 10 % Triton x-100 was added to quench reaction and incubated for 15 minutes at 37 ℃. Chromatin was digested for 2 hours at 37 ℃ with slow rotation by adding 50 μL of 10 x NEB buffer 2 and 15 μL of MboI (NEB R0147M). After heat inactivate MboI by incubating at 62 ℃ for 20 minutes, the DNA ends was marked by biotin using fill-in master mix (37.5 μL 0.4 mM biotin-dATP, 1.5 μL 10 mM dCTP, 1.5 μL 10 mM dGTP, 1.5 μL 10 mM dTTP, 10 μL 5U/μL DNA Polymerase I, Large (Klenow) Fragment) for 1 hour at 37 ℃ with slow rotation. 948 μL of ligation master mix (150 μL 10X NEB T4 DNA ligase buffer with 10 mM ATP, 125 μL 10% Triton X-100, 3 μL 50 mg/mL BSA, 10 μL 400 U/μL T4 DNA Ligase, 660 μL Water) was using to connect proximal DNA ends at room temperature for 4 hours with slow rotation. After centrifuging at 2,500g for 5 minutes, cell pellet was washed once with 1mL ChIP buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl, 1x protease inhibitors), and resuspended using 0.7 mL ChIP buffer with 10 minutes incubation on ice. The cell pellet was sonicated using Bioruptor Plus Sonicator by high intensity, 30 seconds on, 30 seconds rest, 15 cycles. The protein-DNA complexes was pre-clean using 25 μL of protein G beads with incubation at 4 ℃ for 1 hour. The primary antibody was added and incubation at 4 ℃ overnight with slow rotation. Beads washing, DNA elution, and precipitation were the same as ChIP-seq and DNA fragment was dissolved using 10 μL of water. VAHTS Universal DNA Library Prep Kit (Vazyme ND607-01) was used for library construction. After end-repair and adaptor were performed under kit protocols. 10 μL Streptavidin T1 beads were washed twice with Tween washing buffer (5mM Tris-HCl pH7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween 20) and resuspended with 10 μL of 2X Biotin Binding Buffer (10mM Tris-HCl pH7.5, 1mM EDTA, 2M NaCl). The ligated DNA was mixed with streptavidin beads suspension and incubated at room temperature for 15 minutes with slow rotation to enrich biotin labeled DNA. Beads-DNA complexes were washed twice with 500 μL of Tween washing buffer at 55 ℃ for 2 minutes with 1000 rpm shaking. Beads-DNA complexes were washed with 100 μL TE buffer (10mM Tris-HCl pH7.5, 5 mM MgCl2, 10% Dimethylformamide) and resuspended with 20 μL water. The DNA was amplified by PCR (95°C, 3 min; 98°C, 20 s, 60°C, 15 s, 72°C, 30 s for 13 cycles; and a final extension at 72°C, 5 min), and the DNA fragments were select by AMPure XP beads (0.7 x and 0.3 x). The DNA was dissolved using 20 μL H2O.