Fig2c gH2AX and SYCP3 co-staining for Prophase I staging in male meiosis

Raw image dataset for gH2AX and SYCP3 co-staining for Prophase I staging in male meiosis for P21, P28 and adult Wdr62 genetrap testis.<br>Please see Becherel, O. J.<i> et al.</i> Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing. <i>PLoS Genet</i> <b>9</b>, e1003435, doi:10.1371/journal.pgen.1003435 (2013) for spermatocyte spread and immunofluorescence protocol. <br>Prophase I staging using gH2AX and SYCP3 co-staining:Pacheco, S.<i> et al.</i> ATR is required to complete meiotic recombination in mice. <i>Nat Commun</i> <b>9</b>, 2622, doi:10.1038/s41467-018-04851-z (2018).<br><br>Testes were decapsulated, chopped and resuspended in DMEM (Gibco) before passing through 100μm cell strainer. Cells were pelleted by centrifuging at 500rpm at room temperature for 5mins and then resuspended in 0.1M sucrose. 10μL of cell suspension was spread onto glass slides pre-wetted in 100% methanol or 4% paraformaldehyde and fixed for 2 hrs. The slides were subsequently washed with PBS and air-dried in the presence of Kodak Photo-Flo 200 (Kodak, 1 in 250 dilution). Slides were stored at -80°C. For immunostaining, slides were rehydrated in PBS and blocked in blocking buffer (20% FBS, 0.2% Triton X in PBS) for 1 hr before incubating with primary antibody at 4°C overnight. The next day, slides were incubated with secondary antibody and counterstained with DAPI before mounting in Prolong Diamond (Invitrogen). The primary and secondary antibodies used are listed in Supplementary Table 1. <br>