GSM6734584: DN3_DpnII_HiC_rep2; Mus musculus; Hi-C
Five million aliquots of cells were fixed in 2% formaldehyde for 10 min, quenched with 125 mM cold glycine, then collected by centrifugation and washed with PBS. Cells were lysed in lysis buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 0.2% NP-40, protease inhibitor cocktail) on ice for 30 min and nuclei collected by centrifugation and resuspended in DpnII restriction buffer. Thymocytes were permeabilized by treatment with 0.8% SDS for 20 min at 65°C, followed by 40 min at 37°C, then with 3.3% Triton X-100 for 1 h at 37°C. Nuclei aliquots were digested overnight at 37°C with 1500 U DpnII, then cohesive ends were filled in with biotin tags by incubating for 90 min at 37°C with 15 μM dATP, 15 μM dTTP, 15 μM dGTP, 15 μM biotin-14-dCTP and 25 U DNA polymerase I Klenow fragment. Nuclei were collected by centrifugation and in situ ligation was performed overnight at 16°C in 500 μL volumes of 1X ligase buffer (NEB) with 20,000 U T4 DNA ligase, before de-crosslinking overnight at 65°C with 0.75 mg/mL proteinase K. DNA was purified by RNase A treatment, phenol/chloroform extraction and isopropanol precipitation. Five microgram aliquots of DNA were sonicated to a fragment size of ~100-400 bp in a Covaris sonicator E220 and bound to Steptavidin MyOne T1 beads, following the manufacturer's instructions. The DNA was then end-repaired by incubating for 1 h at 20°C with 0.8 mM dNTPs, 0.3 U/μL T4 DNA polymerase, 1 U/μL T4 polynucleotide kinase and 0.1 U/μL DNA polymerase I Klenow fragment in ligase buffer, then A-tailed by incubating for 1 h at 37°C with 200 μM dATP and 0.2 U/μL Klenow fragment (3'-5' exo-) in NEBuffer 2 (NEB), then Illumina PE adapter was added by incubating overnight at 20°C with 15 μM PE adapter and 2000 U T4 DNA ligase in ligase buffer, washing the beads with appropriate buffers in between each treatment. Hi-C material was then amplified from the beads with 7-9 cycles of PCR using Herculase II Fusion DNA polymerase (Agilent) according to the manufacturer's instructions. Products resulting from extract protocol are immediately ready for cluster generation on Illumina HiSeq 4000, following manufacturer's instructions.