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.