AbstractQuantitative analysis of gene expression is crucial for understanding the molecular mechanisms underlying genome regulation. RNA‐seq is a powerful platform for comprehensive investigation of the transcriptome. In this unit, we present a general bioinformatics workflow for the quantitative analysis of RNA‐seq data and describe a few current publicly available computational tools applicable at various steps of this workflow. These tools comprise a pipeline for quality assessment and quantitation of RNA‐seq data that starts from raw sequencing files and is focused on the identification and analysis of genes that are differentially expressed between biological conditions. © 2018 by John Wiley & Sons, Inc.