RNA-Seq Experiment and Data Analysis.

With the ability to obtain several millions of reads per sample, high-throughput RNA sequencing (RNA-Seq) enables investigation of any transcriptome at a fine resolution. Not just the messenger RNA (mRNA), but a wide variety of different RNA populations (e.g., total RNA, microRNA, long ncRNA, pre-mRNA) can also be investigated using RNA-Seq. While facilitating accurate quantification of gene expression, RNA-Seq offers the opportunity to estimate abundance of isoforms and find novel transcripts and allele-specific transcripts. In this chapter, we describe a protocol to construct an RNA-Seq library for sequencing on Illumina NGS platforms and a computational pipeline to perform RNA-Seq data analysis. The protocols described in this chapter can be applied to the analysis of differential gene expression in control versus 17β-estradiol treatment of in vivo or in vitro systems.