A complete bioinformatic protocol for analysis of RNA-Seq data using our tools has been published at Nature Protocols. The protocol covers read alignment with TopHat, gene and transcript discovery with Cufflinks, annotation analysis with Cuffmerge and Cuffcompare, differential expression analysis with Cuffdiff, and visualization with CummeRbund. Several variants of the protocol are included for those who wish to forgo certain analysis steps, such as gene discovery.