This is a practical, hands-on tutorial designed to give participants experience with RNA-Seq data analysis using Tophat and Cufflinks in Galaxy. The analysis in this tutorial is typical of experiments in eukaryotic species with high-quality genomes and genome annotation available. Participants are expected to be familiar with next-generation sequence data and basic theory of RNA-Seq. Participants do not need previous experience with Tophat or Cufflinks. Prerequisites include: GALAXY 101 or experience with GALAXY and the Galaxy RNA-Seq Lecture.