An undergraduate laboratory on RNA sequencing analysis of bacterial gene expression

Next generation sequencing has revolutionized molecular biology and has provided a mechanism for rapid DNA and RNA sequence analysis. Yet, there are few resources to introduce next generation sequencing into the undergraduate biochemistry and molecular biology curriculum. Herein, we describe the design, execution, and assessment of a four-week laboratory for junior and senior undergraduate students that focuses on bacterial gene expression changes detected by RNA sequencing (RNA-seq). In the laboratory, students analyze a bacterial RNA-seq dataset in detail and answer questions relating to the impact of DNA methylation on bacterial gene expression. In addition, students confirm key results from the RNA-seq dataset using qRT-PCR and compare their results to similar experiments in the literature. A major strength of the laboratory is the ability of students to analyze raw RNA-seq data. In addition, another strength of the laboratory is the utilization of both dry approaches (informatics and statistics) and wet approaches (RNA isolation, cDNA synthesis, and qRT-PCR) to answer bacterial gene expression questions. Assessment of the laboratory indicates that significant learning gains were achieved with respect to next generation sequencing and RNA-seq. We expect that the laboratory will be a valuable resource as is, or via modification with other datasets and projects. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 161-167, 2019.