GSE109195
Comparing the Transcriptomes of Marf1-genetrap (GT) oocytes with those microinjected with mRNAs for wild type MARF1 (GTWT) or D272-mutated MARF1 (GTD272) by RNA-Seq Analysis
The goal of this study is to reveal the globle effect of supplementation with either wild type MARF1or D272-mutated MARF1 on the steady-state levels of mRNAs in Marf1-gene trap GV-stage fully-grown oocytes(FGOs) by comparing the corresponding transcriptomes via RNA-Seq Analysis. Overall design: Marf1-genetrap (GT)  GV-stageFGOs were microinjected with mRNAsencoding wild type MARF1 or D272-mutated MARF1, and cultured for 48 h in milrinone supplemented medium. Then the oocytes 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).