To detect mouse SPO11-oligonucleotide complexes, SPO11 is immunoprecipitated from testis lysates and immunoprecipitates are labeled with terminal transferase (TdT) in the presence of radioactive nucleotides. This protocol is largely derived from the corresponding protocol from budding yeast (Chapter 12 from this volume). Unlike synchronous meiotic cultures of yeast, only a small fraction of cells in adult testes are spermatocytes in early meiotic prophase (1
), the stage in which SPO11-oligonucleotide complexes are likely to be present based on results from budding yeast (2
). In addition, the amount of SPO11-oligonucleotide complexes is likely to be comparable per meiotic cell in mouse and budding yeast due to similar numbers of SPO11-induced double-strand breaks (3
), but the mouse genome (~2.5 × 109
bp) is about 200 times the size of the budding yeast genome (~1.2× 107
bp). The small amount of SPO11-oligonucleotide complexes versus the large amount of genomic DNA in mouse poses a challenge for detection. To increase the signal to noise ratio, a simple step of ultracentrifugation is used to pellet most genomic DNA from crude testis lysates, and SPO11-associated oligonucleotide complexes are then immunoprecipitated from the supernatant. To determine the size of SPO11-associated oligonucleotides, SPO11-oligonucleotide complexes are purified using SDS-PAGE and then deproteinized. DNA is then precipitated and resolved on a sequencing gel.