RNA-seq uses next-generation sequencing technology to determine the transcription profile of an organism in a quantitative manner. With respect to microarrays, this methodology allows greater resolution, increased dynamic range, and identification of new features such as previously unannotated genes and noncoding RNAs. Here we describe how to extract RNA from mycobacterial cultures, how to prepare libraries for Illumina sequencing, and the bioinformatics analysis of the sequencing data to determine the transcription profile.