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.Please see Becherel, O. J. et al. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing. PLoS Genet 9, e1003435, doi:10.1371/journal.pgen.1003435 (2013) for spermatocyte spread and immunofluorescence protocol. Prophase I staging using gH2AX and SYCP3 co-staining:Pacheco, S. et al. ATR is required to complete meiotic recombination in mice. Nat Commun 9, 2622, doi:10.1038/s41467-018-04851-z (2018).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.

本数据集为针对P21、P28及成年Wdr62基因陷阱雄性小鼠睾丸的雄性减数分裂I前期分期实验的gH2AX（磷酸化组蛋白H2AX）与SYCP3（联会复合体蛋白3）共染色原始图像数据集。相关精母细胞铺片及免疫荧光染色实验方案可参考Becherel OJ等人2013年发表于《PLOS Genetics》的研究：Senataxin与DNA损伤应答蛋白在减数分裂重组及基因沉默中发挥关键作用，PLoS Genet 9, e1003435, doi:10.1371/journal.pgen.1003435。采用gH2AX与SYCP3共染色进行减数分裂I前期分期的方法，可参考Pacheco S等人2018年发表于《Nature Communications》的研究：ATR蛋白是小鼠完成减数分裂重组所必需的，Nat Commun 9, 2622, doi:10.1038/s41467-018-04851-z。实验样品制备流程如下：取出睾丸并剥离被膜，剪碎后重悬于DMEM培养基（Gibco），随后通过100μm细胞筛过滤。将滤液以室温500rpm离心5分钟收集细胞沉淀，重悬于0.1M蔗糖溶液中。取10μL细胞悬液滴加至预先用100%甲醇或4%多聚甲醛润湿的载玻片上，固定2小时。随后用PBS洗涤载玻片，并用经250倍稀释的Kodak Photo-Flo 200（柯达公司）浸润后自然晾干。制备好的载玻片保存于-80℃冰箱。免疫荧光染色步骤：先将载玻片于PBS中复水，随后用封闭缓冲液（含20%胎牛血清、0.2% Triton X的PBS溶液）封闭1小时，再于4℃下与一抗共同孵育过夜。次日，用二抗孵育载玻片，并用DAPI进行复染，最后用Prolong Diamond封片剂（Invitrogen）封片。本实验所用一抗与二抗信息详见补充表1。