GSM5211786: pEF-1-D; Sus scrofa; Hi-C
The 1×10^6 cells were cross-linked with a final concentration of 4% formaldehyde for 30min at room temperature and followed by quenching with glycine in a final concentration of 0.25 mol/L. Mixtures were next centrifuged at 1,500×g for 10min at room temperature, and separated supernatant were added lysis buffer and incubate 15 min on ice. The mixture was then centrifuged at 5,000×g for 10 min in room temperature. The sediment was washed with 100μL 1×NEBuffer 2. The mixture was added with SDS in a final concentration of 0.1% and incubate 10min at 65°C, then added with TritonX-100 in a final concentration of 1% and incubate 15min at 37°C, thus nuclei of cells were permeabilized. DNA was digested with 200 units of DpnII (a 4-cutter restriction enzyme) for one hour at 37°C. The restriction fragment overhangs were filled and labeled by biotinylated nucleotides and then ligated in a small volume. After crosslink reversal,  DNA were purified and sonicated to fragments about 300-500 bp by applying Covaris S220 sonicator, at which point ligated fragments were pulled down with Dynabeads™ M-280 Streptavidin (Invitrogen, Cat.N: 11206D) and also were end repaired and A-tailed. Adaptors were next ligated and DNA fragments were PCR amplified using KAPA Hyper Prep Kit (Roche, Cat.N: KK8504) for 8-10 cycles. These fragments were then performed double-size selected using AMPure XP Beads (Beckman, Cat.N: A63882) in order to isolate fragments between 300 and 800bp, which were prepped for sequencing at BGISEQ-500 platform to provided 100 bp paired-end reads. Consequently, we generated a total of ~10.91-billion valid contacts of 16 Hi-C libraries (~681.95 M contacts per library) for pgEpiSCs, and a total of ~14.34-billion valid contacts of 16 Hi-C libraries (~895.96 M contacts per library) for pEFs.