Transcriptome analysis in C.elegans from Parental P0 to F3 generations under hypoxic stress in both WT and hrde-1 mutant [RNA-seq]

The goal of RNA-seq is to identify the differentially expressed genes in the wild-type (WT) and hrde-1 mutant of C.elegans at the normoxia reared and hypoxia treatment conditions at P0 generation, and those maintained dysregulated genes in F1, F2 and F3 generation. Two biological replicates were assigned for each group and in total 32 groups were prepared for RNA-seq libraries with 500 ng mRNA as starting materials using NEXTflex Illumina qRNA-Seq Library Prep Kit (Bioo Scientific). We mapped about 20 - 30 million reads per sample to C.elegans (WS235) with RSEM pipline. Differential analysis were conducted using EdgeR. The significant genes were conducted by computing NB exact genewise tests with corrected p < 0.05. Messenger RNA from WT and hrde-1 under normoxia or hypoxia treatment worms from P0 to F3 generations were extacted, and cDNA libraries were generated for deep sequencing, in duplicate, using paired-end 150bp on Illumina Hiseq4000 platform

本RNA测序（RNA-seq）的研究目标为，鉴定常氧与低氧培养条件下P0代秀丽隐杆线虫（Caenorhabditis elegans, C.elegans）野生型（wild-type, WT）与hrde-1突变体中的差异表达基因，以及在F1、F2、F3代持续失调的基因。每组设置2个生物学重复，总计制备32组RNA测序文库，以500 ng信使RNA（mRNA）作为起始材料，使用NEXTflex Illumina qRNA-Seq Library Prep Kit（Bioo Scientific）完成建库。将每个样本约2000万至3000万条reads通过RSEM流程比对至秀丽隐杆线虫WS235版本参考基因组。差异表达分析采用EdgeR工具完成，通过NB精确基因水平检验计算显著差异基因，校正后P值小于0.05即为显著差异基因。提取P0至F3代经常氧或低氧处理的野生型与hrde-1突变体线虫的mRNA，构建cDNA文库以进行深度测序，设置生物学重复两次，采用150bp双端读长在Illumina Hiseq4000测序平台完成测序。