Next-generation RNA-sequencing (RNA-Seq) is rapidly outcompeting microarrays as the technology of choice for whole-transcriptome studies. However, the bioinformatics skills required for RNA-Seq data analysis often pose a significant hurdle for many biologists. Here, we put forward the concepts and considerations that are critical for RNA-Seq data analysis and provide a generic tutorial with example data that outlines the whole pipeline from next-generation sequencing output to quantification of differential gene expression.