RNA‐Seq: A Method for Comprehensive Transcriptome Analysis

A recently developed technique called RNA Sequencing (RNA-Seq) uses massively parallel sequencing to allow transcriptome analyses of genomes at a far higher resolution than is available with Sanger sequencing- and microarray-based methods. In the RNA-Seq method, complementary DNAs (cDNAs) generated from the RNA of interest are directly sequenced using next-generation sequencing technologies. The reads obtained from this can then be aligned to a reference genome in order to construct a whole-genome transcriptome map. RNA-Seq has been used successfully to precisely quantify transcript levels, confirm or revise previously annotated 5' and 3' ends of genes, and map exon/intron boundaries. This unit describes protocols for performing RNA-Seq using the Illumina sequencing platform.