title: GSM5521848: atm het amplicon 4; Mus musculus; OTHER
library_construction_protocol: Based on Cole et at al., 2014 Nature Genetics and Gaysinskaya et al., 2014 Cytometry A, with modifications. DNA was isolated from testicular single-cell suspensions. Testes were decapsulated and seminiferous tubules incubated at 33 ℃ in 10 ml Gey's Balanced Salt Solution (GBSS) with 0.5 mg/ml collagenase and 2 µg/ml DNase I for 15–20 min, shaking at 500 rpm. The tubules were washed twice with 10 ml GBSS and digested with 0.5 mg/ml trypsin in 10 ml GBSS, supplemented with 2 µg/ml DNase I, for 25–40 min at 33 ℃, shaking at 500 rpm. Trypsin was inhibited by adding 10% fetal calf serum. Cell suspension was obtained by repeated pipetting for 2 min and filtration through a 70-µm cell strainer. All of the mutant cell suspension and one-third to one-half of the wild-type cell suspension was transferred to a 15 ml tube and centrifuged for 3 min at 1550 x g. The cell pellet was resuspended by tapping the tube, followed by the addition of 600 µl lysis buffer (200 mM NaCl, 100 mM Tris-Cl pH 7.5, 5 mM EDTA, 0.5% SDS, 0.5 mg/ml Proteinase K). The lysate was transferred to a 1.5 ml Eppendorf tube and incubated overnight at 55 ℃. Genomic DNA was isolated using phenol-chloroform extraction and alcohol precipitation. Four ~580-bp overlapping amplicons of the hotspot on Chr1 were amplified using the following primer pairs: amplicon 1, Chr1_MiSeq_FW1: 5'-TCATGTCCGGTTTGGATGCC-3' and Chr1_MiSeq_RV1: 5'-CGAATCAAAGACTCCAGAAGAGG-3'; amplicon 2, Chr1_MiSeq_FW2: 5'-AGTAGCAAGAAGGGCTGGAG-3' and Chr1_MiSeq_RV2: 5'-AGAGACAGATGTGGAGGCTAAC-3'; amplicon 3, Chr1_MiSeq_FW4: 5'-CTGAATCTCTCCTGGTGTGTC-3' and Chr1_MiSeq_RV4: 5'-AGGACATTGAATGTTTTTATGGTTC-3'; amplicon 4, Chr1_MiSeq_FW5: 5'-AAACAGAAGAAGCAGTAATGCC-3' and Chr1_MiSeq_RV5: 5'-GTGTGCAACTGGGTATATTTCAG-3'. For each amplicon, PCR conditions were: 96 ℃ for 2 min followed by 22 cycles at 96 ℃ for 20 s, 59 ℃ for 30 s and 65 ℃ for 35 s. PCR was performed in a 50 µl total containing 1.5 U Taq DNA polymerase and 0.3 U Pfu Turbo DNA polymerase in the Jeffreys buffer (Cole et al., 2010 Molecular Cell) with 50 ng of testis genomic DNA. Testis DNA was derived from 3-5 different mice for each genotype and amplified independently. PCRs were carried out in 96-well plates. For each amplicon, 576 PCRs were performed with a total of 28.8 µg genomic DNA, equivalent to 9.6 million haploid genomes. PCRs were pooled, and a 1200-µl aliquot was purified and concentrated on spin columns as follows: For each 100 µl PCR aliquot, 400 µl binding buffer from the kit was added, and then the sample was applied to the column and centrifuged. This step was repeated 6 times in each of two columns before washing and elution with 37 µl elution buffer. Eluted DNA was combined from the two columns and a 15-µl aliquot was run on a 2% agarose gel to estimate DNA concentration, which was ~2 ng/µl. 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.