Comparing the Transcriptomes of Marf1 wildtype (WT) oocytes with those of Marf1-genetrap (GT-Mut) and Marf1-D272 mutant(D272-Mut) oocytes by RNA-Seq Analysis

The goal of this study is to reveal the globle changes of oocyte transcriptome after mutations of Marf1 either by gene trap or knockin of point mutation by comparing the corresponding transcriptomes via RNA-Seq Analysis. Marf1 wildtype (WT), Marf1-genetrap (GT-Mut), and Marf1-D272 mutant(D272-Mut) GV-stageFGOs were collected in RLT lysis buffer for RNA-Seq analysis. 4 replictates of each treatment, with 80 oocyte per samples, were collected for the experiment. Total RNA was extracted with RNeasy Micro Kit (Qiagen, Germantown, MD, USA) according to manufacturer's instructions, and the mRNA library was constructed using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA) according to the instruction manual, which includes sequential RNA fragmentation, reverse transcription using random primers, second strand cDNA synthesis, end repair, dA-tailing, adapter ligation, U excision, and PCR enrichment. The oocyte mRNA 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).