DSB landscape mature human spermatozoa
Two distinct cell types were used for the breakome analysis, namely isolated sperm heads and cultured IMR90 cells. The BLISS experiments were performed , with slight modifications adjusted to the characteristics of mature spermatozoa (Yan et al., 2017; Bouwman et al., 2020). Briefly, isolated sperm heads from a total ejaculate or roughly 2x106 cultured IMR90 cells were fixated in LMP agarose and lysated using DTT and proteinase K. Genomic DNA was blunted, Agarose was digested and the adapter containing the RNA polymerase promoter was ligated. Unligated adapters were removed using gel electrophoresis and the isolated gDNA was fragmented for in vitro transcription. The resulting RNA was polyadenylated, reverse transcribed using the Biozym cDNA. Libraries were prepared using the NEBNext Ultra II DNA PCR-free Library Prep Kit for Illumina and sequenced employing a PE150 strategy on a NovaSeq6000.