Comparing the Transcriptomes of WT oocytes with Lsm14b KO oocytes by RNA-Seq Analysis

The success of human reproduction relies on high quality oocytes. Oocyte quality is manifested by the competence to complete meiosis, to be fertilized, and to support embryonic development. This meiotic and developmental competence is gradually established during the course of oocyte and follicle development, and is determined in large part by the autonomous gene expression program intrinsic to the oocyte. In order to explore the regulatory role of LSM14B in oocyte, we analyzed the effect of Lsm14b KO on gene expression in GV-stage fully-grown oocytes (FGOs) in mice by comparing the corresponding transcriptomes via RNA-Seq Analysis. Overall design: WT and Lsm14b KO GV-stage FGOs were collected in lysis buffer to release all RNAs, which then 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, with 20 oocyte per samples, 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).