Conduct and Quality Control of Differential Gene Expression Analysis Using High-Throughput Transcriptome Sequencing (RNASeq).

High-throughput transcriptome sequencing (RNASeq) represents one of the most comprehensive and scalable methods to analyze global gene expression. It allows for absolute quantification of gene expression and also enables the discovery of novel transcripts and alternatively spliced isoforms. This chapter provides hand-on tools and a step-by-step procedure to analyze RNASeq data from punctures of two different retinal tissues (retina and RPE-choroid-sclera) at two different locations (periphery and macular region) from eight individuals. The procedure described in this chapter will use various programs from the free, open-source Tuxedo Suite software package to analyze sequencing data and to ascertain genes that are differentially expressed between retina and RPE-choroid-sclera.