RNA-sequencing (RNA-seq) has become the method of choice for whole transcriptome analysis, as it enables profiling of a wide range of RNA molecules and the always evolving bioinformatic pipelines allow the extraction of diverse biological information from a single sample. However, there is not a gold-standard RNA-seq protocol nor an optimal bioinformatic pipeline defined, which can challenge new users. The aim of this chapter is to describe the basic RNA-sequencing pipeline, from RNA extraction and library preparation to the bioinformatic preprocessing and downstream analyses. The steps are oriented to the transcriptome analysis of the skin of TR-deficient mice, but the protocol can be easily translated to other tissue types or organisms.