GSM9708458: mre11_null_amplicon_3; Mus musculus; OTHER
DNA was isolated from testicular single-cell suspensions according to the previously published protocol (Lukaszewicz et al., 2021 Cell). Mre11 conditional knockout mice, here referred to as Mre11–/–, were used (Buis et al., 2008 Cell; Kim et al., 2025 Nat Commun). Generation of amplicons and deep sequencing were performed as previously described (Lukaszewicz et al., 2021 Cell). Briefly, four overlapping ~580-bp amplicons of the Chr1 single hotspot were amplified using published primers (Lukaszewicz et al., 2021 Cell). PCRs were carried out in 96-well plates in 50 µl reactions, each containing 50 ng input genomic DNA, 1.5 U Taq DNA polymerase, and 0.3 U Pfu Turbo DNA polymerase in the in the Jeffreys buffer (Cole et al., 2010 Molecular Cell). For each amplicon, PCR conditions were the same: denaturation (2 min at 96°C), followed by 22 cycles of amplification (20 s at 96°C, 30 s at 59°C, and 35 s at 65°C). Testis DNA was isolated from three Mre11–/– mice and amplified independently. A total of 576 PCRs were performed for each amplicon, with input DNA equivalent to 9.6 million haploid genomes. Next, for each amplicon, PCR products were pooled, and a 1200-ml aliquot was purified and concentrated on spin columns (Lukaszewicz et al., 2021 Cell). Approximately 60 µl of DNA at a concentration of 1–2 ng/µl was used for library preparation and deep sequencing. The library was prepared by ligation of indexed adapter sequences using the KAPA HyperPrep Kit (Roche). Sequencing was performed using Illumina MiSeq (2 x 300-bp paired-end reads). In a single run, libraries from amplicons 1–4 were mixed with 5% PhiX DNA control library.