GSM4907506: DKO2 (HTGTS); Mus musculus; OTHER
Genomic DNA was sonicated with 2 pulses of 5 secs and and 1 minute off using the Bioruptor pico sonicator (Diagenode). LAM-PCR was performed using Phusion High-Fidelity DNA Polymerase (Thermo Fisher) and Sμ 5' biotinylated bait (TAGTAAGCGAGGCTCTAAAAAGCA). 4 independent PCRs were run with ~2.5 μg of sonicated DNA each, with 90 cycles of primer elongation. The biotinylated DNA fragments were purified using Dynabeads T1 streptavidin beads (Invitrogen) at room temperature, on a rotisserie, overnight. On bead ligation was performed with T4 DNA ligase (NEB) and annealed bridge adaptors (GCGACTATAGGGCACGCGTGGNNNNNN-NH2 and 5'P-CCACGCGTGCCCTATAGTCGC-NH2) for 1h at 25°C, 2h at 22°C, and overnight at 16°C. 15 cycles of nested PCR were performed on the DNA-bead complexes using an Sμ nested primer (ACACTCTTTCCCTACACGACGCTCTTCCGATCT-GGTAAGCAAAGCTGGGCTTG) and a reverse universal primer I7 (GACTGGAGTTCAGACGTGTGCTCTTCCGATCT-GACTATAGGGCACGCGTGG). The bold regions represent the complementary regions to the genomic sequence in the Sμ nested primer and the bridge adaptor for I7 primer. PCR products were cleaned with the QIAquick Gel Extraction Kit (Qiagen) and the final barcoding/indexing PCR was performed using the P5-I5 and P7-I7 universal dual indexing primers (NEB) for 15 cycles and purified using Ampure beads purification with a cutoff of 200 bp. Library quality was evaluated by Bioanalyzer (Agilent) and sequencing performed using the Illumina Miseq Reagent Kit v2 (600 cycles) following the manufacturer's instructions. DNA junctions from 2 independent experiments were combined for analyses.