RNA-sequencing (RNA-seq) is a technique that employs next-generation DNA-sequencing technology to simultaneously sequence all of the RNA transcripts in a cell. It can provide valuable insights into transcript and operon structure, and is rapidly replacing expression microarrays as the technique of choice for determining global gene expression profiles in bacteria. Herein we outline the procedures involved in performing RNA-seq with samples of RNA from Staphylococcus aureus. We draw particular attention to key aspects of sample preparation, such as RNA integrity and removal of ribosomal RNA, and provide details of critical steps in downstream data analysis.