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. 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).

本研究旨在通过RNA测序（RNA-Seq）分析对比对应转录组，揭示野生型MARF1或D272突变型MARF1补充处理对Marf1基因陷阱（Marf1-gene trap）GV期完全成熟卵母细胞（Fully-grown oocytes, FGOs）中mRNA稳态水平的全局影响。将Marf1基因陷阱（Marf1-genetrap, GT）GV期FGOs显微注射编码野生型MARF1或D272突变型MARF1的mRNA，随后在添加米力农的培养基中培养48小时。随后将卵母细胞收集于RLT裂解缓冲液中，用于RNA测序分析。本实验每组处理设置4次生物学重复，每个样本包含80枚卵母细胞。按照制造商说明书，使用RNeasy微量试剂盒（Qiagen，美国马里兰州日耳曼敦）提取总RNA；随后参照操作手册，使用适配Illumina平台的NEBNext UltraTM RNA文库制备试剂盒（NEB，美国马萨诸塞州伊普斯维奇）构建mRNA文库，文库构建流程依次为：RNA片段化、随机引物反转录、第二链cDNA合成、末端修复、dA尾加尾、接头连接、U碱基切除及PCR富集。卵母细胞mRNA文库在Illumina HiSeq X Ten测序平台上进行150bp双端测序。所有通过过滤的测序读段（reads）将被修剪，以去除低质量碱基及接头序列。随后使用TopHat2（v2.0.13）将测序读段比对至mm10参考基因组，并使用Cufflinks（v2.2.1）计算并标准化FPKM值。使用Cuffdiff（v2.2.1）的默认参数计算差异表达基因。对所有样本的log₂(FPKM+1)值进行分层聚类分析，聚类所用基因的筛选标准为：最大FPKM值≥1，且log₂(FPKM+1)的标准差位列前10%。