RNA-Seq examines global gene expression to provide insights into cellular processes, and it can be particularly informative when comparing contrasting physiological states or strains. Although relatively routine in many laboratories, there are many steps involved in performing a transcriptomics experiment to ensure representative and high-quality results are generated for analysis. In this chapter, we present the application of widely used bioinformatic methodologies to assess, trim, and filter RNA-seq reads for quality using FastQC and Trim Galore, respectively. High-quality reads are mapped using Bowtie2 and differentially expressed genes across different groups were estimated using the DEseq2 R-Bioconductor package. In addition, we describe the various steps to perform the sample-wise data quality assessment by generating exploratory plots through the DESeq2 package. Simple steps to calculate the significant differentially expressed genes, up- and down-regulated genes, and exporting the data and images are also included. A Venn diagram is a useful method to compare the differentially expressed genes across various comparisons and steps to generate the Venn diagram from DESeq2 results are provided. Finally, the output from DESeq2 is compared to published results from EdgeR. The Clostridium autoethanogenum data are published and publicly available.