RNA sequencing (RNA-Seq) is a powerful tool that captures information about how organisms respond to stimuli in their environment at the molecular level. A common RNA-Seq approach involves isolating and sequencing all of the messenger RNA (mRNA) in a tissue sample taken from an organism. Researchers can compare patterns observed in RNA-Seq data to understand how individuals respond to the environment over minutes, hours, or days and how populations evolve in response to the environment over millions of years. The materials in this repository will guide users through an analysis of RNA-Seq data collected from two California populations of a copepod crustacean, Tigriopus californicus, that were exposed to different levels of salinity. Users will examine the contents of a fastq file that contains raw RNA-Seq data, determine the quality of the RNA-Seq data using a web-server, and test for significant differences in gene expression between the copepod populations using the R packages DESeq2, edgeR, and VennDiagram. The course materials were developed for the 2022 LSU Bioinformatics Workshop held at Louisiana State University, Baton Rouge, LA on July 17-22, hosted by Melissa DeBiasse and Morgan Kelly.