GSM7885794: CD8T,SCA,Donor7; Homo sapiens; Hi-C
Briefly, 5 million cells were cross-linked using 1% formaldehyde for 10 min at room temperature, quenched with 0.125M glycine for 10 min, and were washed with cold PBS. Cross-linked cells were resuspended in 1mL iced-cold Hi-C lysis buffer (10 mM Tris-HCl, pH 8.0; 10mM NaCl, 0.2% NP-40 and protease inhibitor cocktail), and rotated at 4°C for 30 min. The nuclei were resuspended and permeabilized in 0.5% SDS at 62°C for 10 min, quenched by Triton X-100. Subsequently, chromatin was digested by MboI at 37°C overnight (NEB, R0147M). DNA ends were filled by DNA polymerase I (Klenow, NEB, M0210) to incorporate biotin-14-dATP (Jeana Bioscience). Then, blunt end Hi-C DNA ligation was performed using NEB T4 DNA ligase at room temperature for four hours with rotation. After ligation, nuclei were pelleted down and resuspended with Hi-C nuclear lysis buffer (50mM Tris-HCl pH7.5; 10mM EDTA, 1% SDS and protease inhibitor cocktail), and sheared using the parameter of 10/20 s ON/OFF cycle, 25% Amp, 5mins by a QSonica 800R sonicator. We de-crosslinked ~10% of sonicated DNA by heating overnight at 65°C in 1% SDS and 10mM Tris-HCl pH7.5, then treated this with proteinase K, purify biotin-incorporated DNA by C1 streptavidin beads (ThermoFisher). We generated illumina sequencing library using NEB Ultra DNA library kit by following the manufacturer's protocol. Hi-C library was first run on a NextSeq 550 platform with 40nt/40nt mode at a shallow sequencing depth, and then sequenced deeper by a NovaSeq 6000 platform with 150nt/150nt model.