GSM8541633: mESC_BPK25_HiC_rep2_batch2; Mus musculus; Hi-C
Genomic DNA were extracted. Four to five million cells were fixed for 5 min with 2 % formaldehyde in PBS and then the reaction was quenched for another 5 min in 0.125 M glycine. Nuclei were then extracted by incubating cells on ice for 30 min (inverting every 10 min) in 10 mM Tris pH 8.0, 10 mM NaCl, 0.2 % NP-40 (IGEPAL CA-630) and protease inhibitor cocktail (Roche).Hi-C libraries were prepared using an adaptor ligation method. Briefly, biotinylated junctions were captured on streptavidin M-280 dynabeads and digested with DpNII enzyme to generate blunt ends. The blunt ends were A-tailed with Klenow (3'→5' exo-) before ligation with adaptor sequences containing unique barcode. After adaptor ligation DNA was PCR amplified with Illumina primers for 25 cycles and library fragments of ~300-1000 bp (insert plus adaptor and PCR primer sequences) were isolated using AMPure beads (Beckman). The purified DNA was captured on an Illumina flow cell for cluster generation.