RNA-seq is a vital method for understanding gene structure and expression 
patterns. Typical RNA-seq analysis protocols use sequencing reads of length 50 
to 150 nucleotides for alignment to the reference genome and assembly of 
transcripts. The resultant transcripts are quantified and used for differential 
expression and visualization. Existing tools and protocols for RNA-seq are vast 
and diverse; given their differences in performance, it is critical to select an 
analysis protocol that is scalable, accurate, and easy to use. Tuxedo, a popular 
alignment-based protocol for RNA-seq analysis, has been updated with HISAT2, 
StringTie, StringTie-merge, and Ballgown, and the updated protocol outperforms 
its predecessor. Similarly, new pseudo-alignment-based protocols like Kallisto 
and Sleuth reduce runtime and improve performance. However, these tools are 
challenging for researchers lacking command-line experience. Here, we describe 
two new RNA-seq analysis protocols, in which all tools are deployed on CyVerse 
Cyberinfrastructure with user-friendly graphical user interfaces, and validate 
their performance using plant RNA-seq data. © 2018 by John Wiley & Sons, Inc.