GSM3604075: si53BP1_rep2; Homo sapiens; OTHER
Genomic DNA was extracted with proteinase K digestion overnight and phenol/chloroforme extraction. Briefly, DNA was purified and sonicated (Bioruptor, Diagenode) into 500–1,000 bp fragments. LAM-PCR was performed using bait primer coupled with biotin, 1U Phusion polymerase (Thermo Scientific) and the following cycles: 1x (98 °C for 120 s); 80x (95 °C for 30 s; 58 °C for 30 s; 72 °C for 90 s); and 1x (72 °C for 360 s). Biotinylated PCR fragments were incubated with MyOne streptavidin C1 beads (Invitrogen) and rotated for 4 h in 1 M NaCl and 5 mM EDTA buffer at room temperature. After washes with B&W buffer (1 M NaCl; 5 mM Tris-HCl, 0.5 mM EDTA), on-bead ligation was performed using 2.5 mM bridge adaptor, 1 mM hexamine cobalt chloride (Sigma), 15 U T4 DNA ligase (Promega), 15% PEG-8000 (Sigma) and the following cycles: 25 °C for 1 h; 22°C for 2 h; and 16°C O/N. After washing three times with B&W buffer (1 M NaCl, 5 mM Tris-HCl (pH 7.4) and 0.5 mM EDTA (pH 8.0)), the on-bead ligated products were subjected to nested PCR using Phusion polymerase, locus-specific and adaptor primers and the following cycles: 1x (95 °C for 300 s); 15x (95 °C for 60 s; 72 °C 60 s); and 1x (72 °C 600 s). Blocking digestion was then performed with I-SceI for 1 h to remove uncut germline DNA. After purification using a Qiagen column, recovered DNA was PCR-amplified using Illumina primers, Phusion polymerase and the following cycles: 1x (95°C for 180 s); 10x (95°C for 30 s; 60°C for 30 s; 72°C for 60 s); and 1x (72°C for 360 s). The tagged PCR products were size-selected for DNA fragments between 500–1000 bp on a 1% agarose gel and purified using a Qiagen column before loading onto an Illumina Miseq machine for paired-end 2X250 bp sequencing.
GEO Accession
GSM3604075