Identifying the Translatome of WT and Lsm14b KO GV-Stage Oocytes in mice via Scarce Sample Polysome Profiling (SSP-profiling)

RNA-binding proteins (RBPs) have essential functions during oocyte development.In order to explore the regulatory role of RNA-binding protein LSM14B in oocyte, we Identified the translatome of WT and Lsm14b KO GV-Stage Oocytes in mice via Scarce Sample Polysome Profiling (SSP-profiling). Overall design: Germinal vesicle (GV) oocytes were obtained from 3 weeks old WT and Lsm14b KO mice, 46 h after injection with 5 IU pregnant mare serum gonadotropin (PMSG). GV oocytes were then transferred to polyvinyl alcohol (PVA) supplemented with cycloheximide (0.1 mg/mL) and stored at –80? in 1.5 ml tubes. Approximately 1500 oocytes were lysed with MCB Buffer (100mM KCl, 0.1% Triton X-100 50mM HEPES, 2mM MgCl2, 10% Glycerol,1 mM DTT, 20 U/mL RNase inhibitor, 10 mg/mL cycloheximide, plus mini-EDTA-free protease inhibitor cocktai). Oocyte lysates were centrifuged at 12,000g for 10 min at 4°C. Supernatants were loaded on a 10-mL 15%–55% sucrose gradient,centrifuged at 38000rpm for 3 h at 4?(XPN-100,Beckman-Coulter)and collected in 28 fractions, 1ml per fraction. RNAs from the polysome(11-28 fractions) or RNP fractions(1-10 fractions) were precipitated with ethanol overnight and purified with RNeasy Plus Micro kit (Qiagen). Polysome-bound RNAs and RNAs in the RNP fractions were reverse transcribed into first cDNA strands by SuperScript II reverse transcriptase(Invitrogen) and random primers. These cDNAs were amplificated according to Smart-seq2 procedures for construct Illumina sequencing library. 3 replictates of each treatment were collected for the experiment. The library was constructed using KAPA Hyper Prep Kit for Illumina (Roche) according to the instruction manual, which includes end repair, dA-tailing, adapter ligation, post-ligation cleanup, library amplification and post-amplification cleanup. The libraries were sequenced on an Illumina HiSeq X Ten platform with 150bp pair-end reads. All reads passed filter were trimmed to remove low-quality bases and adaptor sequences. Reads were then aligned to the mm10 reference genome using tophat2 (v2.0.13), and FPKMs were calculated and normalized using cufflinks (v2.2.1). The differentially expressed genes were calculated using default parameter of cuffdiff (v2.2.1). Hierarchical clustering was carried on log2(FPKM+1) across samples. Genes used for clustering were selected by maximum FPKM=1 and with top 10% standard deviation of log2(FPKM+1).