RNA exosome complex protects mouse oogenesis by clearing pervasive transcripts and regulating H3K27me3

We performed mouse single oocyte RNA-seq and bulk oocyte CUT&Tag assays in the current project. In details, SMART-seq based single oocyte RNA-seq was performed at adult GV stage, GV3h stage and MII stage, using control and Dis3 oocyte-speicfic knockout (cKO) oocytes. RiboMinus-seq based single oocyte RNA-seq was performed at adult GV stage using control and Dis3 cKO oocytes, and at p20 GV stage using control, Dis3 cKO, Exosc10 cKO and Dis3/Exosc10 double cKO (dcKO) oocytes. Bulk CUT&Tag of anti-H3K27me3 was done in WT and Dis3 cKO oocytes at adult GV stage, and in WT and Dis3/Exosc10 dcKO oocytes at p20 stage. In addition, CUT&Tag of anti-RNA polymerase II (Ser2+Ser5) was performed in WT and Dis3 cKO oocytes at GV stage. All CUT&Tag experiments share the same rabbit-Igg negative control. All p20 stage oocytes were specified as p20. The non-specified GV oocytes were all adult GV oocytes. In SMART-seq based single oocyte RNA-seq, totally 72 samples were performed, including 24 samples at GV stage, 24 samples at GV3h stage and 24 samples at MII stage. At each stage, 8 samples were from control group and 16 samples were from Dis3 cKO group. In RiboMinus RNA-seq, all groups (adult-GV-ctr, adult-GV-Dis3cKO, p20-ctr, p20-Dis3cKO, p20-Exosc10, p20-dcKO) have 8 samples each. In CUT&Tag assays, each group has 3 replicates in totall. The CUT&Tag sequencing results were all firstly randomly sampled for 3M reads for downstream analysis (without deduplication).本研究项目开展了小鼠单个卵母细胞RNA测序（RNA-seq）与批量卵母细胞CUT&Tag实验。具体而言，基于SMART-seq的单个卵母细胞RNA-seq实验覆盖成年卵母细胞GV期、GV3h期与MII期，实验材料为野生型对照与Dis3卵母细胞特异性条件性敲除（cKO）卵母细胞。基于RiboMinus-seq的单个卵母细胞RNA-seq实验分为两部分：成年GV期样本使用野生型对照与Dis3 cKO卵母细胞，p20 GV期样本则使用野生型对照、Dis3 cKO、Exosc10 cKO以及Dis3/Exosc10双条件性敲除（dcKO）卵母细胞。针对抗H3K27me3抗体的批量CUT&Tag实验，分别在成年GV期的野生型与Dis3 cKO卵母细胞，以及p20期的野生型与Dis3/Exosc10 dcKO卵母细胞中完成。此外，本研究还针对抗RNA聚合酶II（Ser2+Ser5）抗体开展了CUT&Tag实验，实验材料为GV期的野生型与Dis3 cKO卵母细胞。所有CUT&Tag实验均采用同一批兔IgG阴性对照。未标注来源的GV卵母细胞均为成年GV卵母细胞。基于SMART-seq的单个卵母细胞RNA-seq实验共包含72个样本，其中GV期、GV3h期与MII期各24个；每个阶段的样本中，对照组与Dis3 cKO组分别包含8个与16个样本。基于RiboMinus-seq的单个卵母细胞RNA-seq实验中，所有实验组（成年GV期对照组、成年GV期Dis3 cKO组、p20 GV期对照组、p20 GV期Dis3 cKO组、p20 GV期Exosc10 cKO组、p20 GV期dcKO组）每组均包含8个样本。CUT&Tag实验每组均设置3个生物学重复。所有CUT&Tag测序数据均首先进行随机抽样，抽取300万条reads用于后续分析（未进行去重处理）。