Transcriptome profiling of Embryonic day 9.5 WT and Clpex mutant mice

Purpose: To determine the differentially expressed genes in the whole E9.5 Clpex mutant embryo, a hypomorph of Pgap2 of the glycosylphosphatidylinositol biosynthetic pathway. 5 WT and 5 Clpex mutant E9.5 embryos were snap frozen on dry ice. RNA was isolated and pooled samples of each genotype were used for paired-end bulk-RNA sequencing (BGI-Americas, Cambridge, MA). RNA-Seq analysis pipeline steps were performed using CSBB [Computational Suite for Bioinformaticians and Biologists: https://github.com/csbbcompbio/CSBB-v3.0]. CSBBÂ Â has multiple modules, RNA-Seq module is focused on carrying out analysis steps on sequencing data, which comprises of quality check, alignment, quantification and generating mapped read visualization files. Quality check of the sequencing reads was performed using FASTQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc). RNA-Seq reads for the mutant and wildtype were paired-end and had ~43 and ~31 million reads respectively.Differential expression analysis was carried out by EBSeq [https://www.biostat.wisc.edu/~kendzior/EBSEQ/]. Differential transcripts are filtered based on LogFC and p-value. Filtered DE transcripts are used for functional and pathway enrichment using toppgene [https://toppgene.cchmc.org/]研究目的：鉴定糖基磷脂酰肌醇（glycosylphosphatidylinositol, GPI）生物合成通路中Pgap2的低功能突变体Clpex突变的E9.5期全胚胎中的差异表达基因。收集5只野生型（WT）与5只Clpex突变型E9.5期小鼠胚胎，经干冰快速冷冻后提取总RNA；将每种基因型的样本分别混合，采用双末端批量RNA测序（BGI-Americas，马萨诸塞州剑桥市）进行测序。测序分析流程通过CSBB[生物信息学工作者与生物学家计算套件：https://github.com/csbbcompbio/CSBB-v3.0]完成。CSBB包含多个功能模块，其中RNA测序模块专注于测序数据的分析流程，涵盖质量控制、序列比对、转录本定量以及生成比对读段可视化文件。测序读段的质量控制使用FASTQC（http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc）完成。突变型与野生型样本的RNA测序读段均为双末端格式，各自的读段数约为4300万与3100万。差异表达分析通过EBSeq（https://www.biostat.wisc.edu/~kendzior/EBSEQ/）完成。差异转录本根据对数倍数变化（LogFC）与p值进行筛选，筛选得到的差异表达（Differential Expression, DE）转录本将通过toppgene（https://toppgene.cchmc.org/）进行功能富集与通路富集分析。