GSM5425806: HiC_ESC_DMSO_rep1; Mus musculus; Hi-C
Three million cells per sample were fixed in suspension with 1 % formaldehyde for 10 minutes and quenched with 0.125 M glycine for 5 minutes in rotation. Cells were washed with PBS and lysed in Hi-C lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Igepal CA-630, 1.15 % Triton X-100 and 1x protease inhibitors (Roche)) for 10 minutes on ice. Nuclei were washed with cold PBS, NEBuffer 2 (NEB) and resuspended in 50 µl of 0.5 % SDS, incubated in 62 °C for 7 minutes and SDS was quenched with 145 µl water and 25 µl 10 % Triton X-100 followed by 15 min incubation in 37 °C. For restriction enzyme digestion, 25 µl of 10X NEBuffer2 and 250 U of MboI (NEB) were added and samples were incubated overnight in 37 °C, followed by additional 1 hour incubation after adding another 250 U of MboI. Enzymes were inactivated for 20 minutes in 65 °C. Samples were spinned in 5000 g, 200 µl of supernatant was removed and sample was resuspended in 200 µl of 1.2X NEBuffer 2. To fill-in DNA overhangs, 50 µl of fill-in buffer (37.5 µl of 0.4 mM biotin-14-dATP (Thermo Fisher, 19524016), 1.5 µl of dCTP, 1.5 µl of dGTP, 1.5 µl of dTTP, 10 mM each, and 8 µl of 5U/µl DNA PolI Large (Klenow) Fragment (NEB)) was added and the samples were incubated in 37 °C for 90 minutes. For ligation reaction, samples were topped up with 663 µl of water, 120 µl of NEB T4 DNA ligase buffer (NEB), 100 µl of 10 % Triton X-100 and 12 µl of 10 mg/ml BSA (NEB) and 2000 U of T4 DNA ligase (NEB) and incubated overnight in 18 °C in rotation. Crosslinks were reversed with Proteinase K and 0.88 % SDS in 10 mM Tris-HCl (pH 7.5) during 30 min in 55 °C, followed by incubation in 68 °C in presence of 2.3 M NaCl for 2 hours. For library preparation, DNA was extracted from samples with ethanol and sodium acetate precipitations, sheared with Covaris E220 and pulled down with 150 µl of T1 Streptavidin Dynabeads (Thermo Fisher). The end-repair for captured DNA was performed in room temperature for 30 minutes using 100 µl reaction with 0.5 U of T4 Polynucleotide Kinase (NEB), 12 U of T4 DNA polymerase (NEB) 5 U of DNA pol I Large (Klenow) Fragment in NEB T4 DNA ligase buffer with 10 mM ATP and 0.5 mM dNTP. After washing the bead-bound DNA, samples were resuspended to NEBuffer 2 with 0.5 mM dATP and 25 U of Klenow exo minus (NEB) and incubated in 37 °C for 30 minutes. After washing the beads, universal sequencing adapters from NEBnext Ultra DNA Library Kit (NEB) were ligated in NEB Quick ligation reaction buffer according to manufacturer's guidelines. Libraries were amplified for 6 PCR cycles and purified using Ampure XP beads (Beckman Coulter). Libraries were paired-end sequenced with NovaSeq 6000 for 400 million fragments for each library.