GSM7105748: Hi-C: HeLa NIPBL depleted G2 rep 1; Homo sapiens; Hi-C
Hi-C sample preparation was performed as described in (Mitter et al, 2020, 2022). All centrifugations were performed at 2500 g, apart from those in the ethanol precipitation step, which were performed at 21000 g. Frozen cell pellets were thawed and then lysed using ice cold Hi-C lysis buffer (10 mM TRIS-HCl pH 8 (Sigma), 10 mM NaCl (Sigma), 0.2% Nonidet P-40 substitute (Sigma), 1 × complete EDTA-free protease inhibitor (Roche) for 30 min at 4 °C. The DNA was then digested using Dpn II (375 U DpnII (NEB, R0543 T/M/L) in 1× Dpn II buffer (NEB, B0543S) for 16 h at 37 °C with shaking at 800 rpm on a thermal block. Cells were centrifuged, the supernatant discarded, and fill-in mix added (38 μM biotin-14-dATP (Thermo Fisher Scientific, 19524016), 38 μM dCTP (Thermo Fisher Scientific, R0152), dGTP (Thermo Fisher Scientific, R0162), dTTP (Thermo Fisher Scientific, R0172), 50 U Klenow Polymerase (NEB, M0210L), 1 × NEB 2 buffer, B7002S). Cells were then incubated for 1 h at 37 °C with gentle rotation. Cells were then spun down, the supernatant discarded, ligation mix added (1 × T4 DNA ligase buffer (Thermo Fisher Scientific, B69), 0.1% Triton X-100 (Sigma, X100-100 ML), 100 μg mL-1 BSA (Sigma, A7030-10G), 50 U T4 DNA ligase (Thermo Fisher Scientific, EL0011) before incubation at room temperature for 4 h with gentle rotation. Cells were spun down, the supernatant discarded, and the pellet resuspended in 200 μL PBS. Genomic DNA (gDNA) was then purified using the DNeasy Blood and Tissue Kit (Qiagen, 69506) according to the manufacturer instructions for tissue culture cells. During gDNA purification, after addition of lysis buffer, RNaseA (Qiagen, 19101) and proteinase K (Qiagen, 19131), samples were incubated at 65 °C for 6 h to reverse crosslinks, before continuing with the rest of the protocol. The purified DNA was then sheared on a Covaris S2 instrument (duty cycle 10%, intensity 5.0, cycles/burst 200) for 25 s. The DNA was sheared in Covaris microTUBEs (Covaris, 520045). Size-selection was then performed to isolate fragments of the correct size using AMPure XP beads (Beckmann Coulter, A63881). A double size selection was performed according to manufacturer instructions. In brief 0.8 x sample volume of beads was first added to the beads, the supernatant was then transferred to a new tube and 0.12 x sample volume beads was then added. The final size-selected DNA was resuspended in dH2O. A biotin-pull down was then performed to select biotin-labelled DNA fragments using Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific, 65001), resuspended in biotin binding buffer (5 mM TRIS-HCl pH 7.5 (Sigma), 0.5 mM EDTA (AppliChem, 1103.1000), 1M NaCl (Merck). Beads were incubated with the samples for 1 h at room temperature with gentle rotation before two washes with Tween wash buffer (5 mM TRIS-HCl, pH 7.5 (Sigma), 0.5 mM EDTA (Applichem, 1103.1000), 1M NaCl (Merck), 0.05% Tween20 (Sigma, P1379-250ML). A final wash with dH2O was then performed to remove residual detergent before the library preparation. The beads were then resuspended in dH2O, and library prep performed on the beads using the NEBNext Ultra II DNA library prep kit for Illumina (NEB, E7645L), according to the manufacturer instructions. The beads were then washed 4 x with Tween wash buffer. Elution of DNA from the beads was performed by incubation with 95% formamide (Sigma Aldrich, F9037-100ML), 10 mM EDTA (Applichem) at 65 °C for 2 min. Samples were then ethanol precipitated (80% EtOH (Sigma, 32221-1L / 2.5L), 100 mM sodium acetate pH 5.2 (Sigma Aldrich, 71180-1kg), incubation at -70 °C for > 16 h). 1 μL GlycoBlue (Thermo Fisher Scientific, AM9515) was also added during the precipitation to better visualise the pellet. Samples were spun down, the supernatant removed, washed once with 75% EtOH, spun down once more and then resuspended in dH2O. 4sT was then converted to methyl-cytosine using OsO4 / NH4Cl as described in (Mitter et al, 2020, 2022). EtOH precipitation was then performed once more to the converted samples as described above. Finally, qPCR was performed to amplify libraries using the NEBUltra Ultra II DNA library prep kit for Illumina (NEB, E7645L). The final libraries were purified using AMP Pure XP beads (Beckmann Coulter, A63881) at 0.55 x sample volume, according to the manufacturer instructions.