GSM8174064: Rad50-sKO-Z_END-seq_rep2; Mus musculus; OTHER
Spermatocyte-enrichment testicular cells were embedded into a final concentration of 0.75% agarose and then lysed and digested with proteinase K and RNase. The plugs that contained  3′ ssDNAs at the end of DSBs were then digested with Exonuclease VII (NEB) and Exonuclease T (NEB) to blunt DNA ends. After blunted, A-tailing were added to the free 3′- OH of blunted ends, then ligated with END-seq hairpin adaptor 1. Subsequently, agarose plugs were melted and dialyzed, the released DNA was sonicated to a shear DNA fragment at the length of about 170 bp. Shear DNA fragment were collected and purified, then the DNA pellet was dissolved in TE buffer. Biotinylated adaptor 1-DNA fragment was further isolated by using MyOne Streptavidin Beads. The second end was repaired and added with A-tail, then ligated with END-seq hairpin adaptor 2. Both sides of hairpin were digested by using USER (NEB) and the resulting DNA fragments amplified by PCR for 16 cycles. Post amplication libraries were selected at 200-400 bp in length by QIAquick Gel Extraction Kit.