RNA-seq Analysis of Msx2fl/fl(Msx2 WT) and Le-Cre+/- Msx2fl/fl(Msx2 CKO) Lens Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived lens transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Lens RNA was extracted from Msx2 WT and Msx2 CKO mice at postnatal day 60 (P60) using RNAeasy Animal RNA Isolation Kit. The freshly frozen RNA solution was prepared for RNA sequencing on the same day. The RNA-seq was carried out using Illumina HiSeq sequencing (Novogene Bioinformatics Institute, Beijing, China). The insert size of the library was tested by Agilent 2100 Bioanalyzer to ensure library quality before Illumina HiSeq sequencing. The company used STAR software to compare RNA-seq sequencing data, HTSeq to analyze the quantitative of gene levels, RSEM to analyze transcript levels quantitatively, ClusterProfiler software to analyze differential gene enrichment, rMATS software to analyze alternative splicing, GATK software to analyze mutation sites and SnpEff software to analyze annotation of the variation sites. After mutation site detection and annotation, statistical analysis was done by Ts/Tv and Hom/Het, respectively. Results: RNA-sequencing results showed that 1,911 differentially expressed genes (DEGs) were detected. Among them, the expression of 1,586 genes were upregulated and the rest downregulated. Lens mRNA profiles of 60-day old Msx2 WT and Msx2 CKO mice were generated by deep sequencing, in once, using Illumina HiSeq sequencing.【研究目的】下一代测序（Next-generation sequencing, NGS）已彻底革新了细胞通路的系统生物学分析。本研究旨在对比基于NGS的晶状体转录组谱分析（RNA-seq）与微阵列、定量反转录聚合酶链反应（qRT-PCR）三种技术方法，并筛选出适用于最优高通量数据分析的实验方案。 【实验方法】本研究于小鼠出生后第60天（P60），从Msx2野生型（Wild Type, WT）及Msx2条件性敲除（Conditional Knockout, CKO）小鼠的晶状体组织中提取总RNA，所用试剂盒为RNAeasy动物RNA提取试剂盒。当日即使用新鲜冻存的RNA样本制备测序文库，用于RNA测序。RNA测序工作由中国北京诺禾致源生物信息学研究所（Novogene Bioinformatics Institute, Beijing, China）依托Illumina HiSeq测序平台完成。在开展Illumina HiSeq测序前，通过Agilent 2100生物分析仪检测文库插入片段大小，以验证文库质量。该机构采用STAR软件完成RNA测序数据的比对分析，使用HTSeq进行基因表达水平定量、RSEM进行转录本表达水平定量、ClusterProfiler软件开展差异基因富集分析、rMATS软件分析可变剪接事件、GATK软件检测突变位点，同时通过SnpEff软件对变异位点进行功能注释。完成突变位点检测与注释后，分别通过转换/颠换比（Ts/Tv）与纯合/杂合比例（Hom/Het）进行统计学检验。 【实验结果】RNA测序结果显示，共检测到1911个差异表达基因（Differentially Expressed Genes, DEGs），其中1586个基因表达上调，剩余基因均表达下调。本研究通过Illumina HiSeq测序平台进行单次深度测序，成功获得了60日龄Msx2野生型及Msx2条件性敲除小鼠的晶状体mRNA表达谱。