Deep sequencing analysis of gene expression (DSAGE) measures global gene transcript levels from only 1 to 2 µg total RNA by massively parallel sequencing of cDNA tags. This unit describes the construction of 21‐bp cDNA tag libraries appropriate for massively parallel sequencing and analysis of the resulting sequence data. The adapter oligonucleotides used are optimized for sequencing with current Illumina massively parallel sequencers, and a step‐by‐step implementation of the analysis protocol is described. The expression profiles obtained are highly reproducible, enabling sensitive detection of differences between experimental conditions as well as assessment of the relative transcript abundance of different genes. Curr. Protoc. Mol. Biol. 93:25B.9.1‐25B.9.16. © 2011 by John Wiley & Sons, Inc.