RNA‐Seq: A Method for Comprehensive Transcriptome Analysis

Abstract 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. Curr. Protoc. Mol. Biol . 89:4.11.1‐4.11.13. © 2010 by John Wiley & Sons, Inc.