GSM6734596: ESC_CRISPRa_Nfatc3_CapHiC_rep1; 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. ESCs were permeabilized by treatment with 0.4% SDS for 1 h at 37°C, followed by 1.6% 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. Material at this stage was quantified on a BioAnalyzer (Agilent) and DpnII and HindIII Hi-C material was sequenced (2x50 nt) on a HiSeq 4000 (Illumina) following the manufacturer's instructions). 750 ng of this DpnII Hi-C material was concentrated in a SpeedVac vacuum concentrator and then target capture was performed using the SureSelect XT Target Enrichment system (Agilent) with the custom-designed library, following the manufacturer's instructions Products resulting from extract protocol are immediately ready for cluster generation on Illumina HiSeq 4000, following manufacturer's instructions.