Short-Read RNA-Seq

Abstract

RNA sequencing (RNA-Seq) has emerged as a powerful and versatile tool for the comprehensive analysis of transcriptomes and has been widely used to investigate gene expression, copy number variation, alternative splicing, and novel transcript discovery. This chapter outlines the methodology for conducting short-read RNA-Seq, starting from RNA enrichment to library preparation and sequencing. Throughout the chapter, practical tips and best practices are provided to guide researchers in order to optimize each step of the RNA-Seq workflow. Multiple quality control steps throughout the workflow that are critical to obtain high-quality RNA-Seq data are also discussed.

Keywords: Library preparation; RNA-Seq; Short-read sequencing.

Fig. 1

RNA Sequencing workflow. Quality control is needed between each major step in RNA Sequencing projects. This review will mostly cover the steps from the QC1 to QC3, focusing on the RNA Library Construction and Sequencing of the Libraries.

Fig. 2

Examples of BioAnalyzer traces with different RNA quality and RINs. High quality RNA samples have RINs over 8–10, and intact 18S and 28S. Partially degraded RNA samples have RINs between 5 and 8, and still have 18S and 28S peaks. Highly degraded RNA (e.g., isolated from FFPE samples) have low RINs below 5 and peaks below or around 200 bp.

Fig. 3

Workflow for Illumina based RNA-Seq library construction. Adapters that contain P7 and P5, the sequences that are required for Illumina flow cell attachment and clustering are attached to the fragmented RNA inserts.