Strategies for Global RNA Sequencing of the Human Pathogen Neisseria gonorrhoeae

Abstract

Over the last several years transcriptomic analysis of bacterial pathogens has become easier and less expensive. This technique is used to determine expression levels for all genes of a particular species or collection of species under a given condition, including genes that are not yet known to exist. While transcriptomics can be a powerful tool to better understand bacterial regulatory responses to specific host environments, the experimental approach and data analysis must be performed correctly to ensure robust, accurate, and translational results. Here, we describe experimental protocols related to transcriptomic analysis of the sexually transmitted disease pathogen Neisseria gonorrhoeae. Methods are described for the extraction of high-quality RNA, examination of RNA to ensure quality, the generation of cDNA libraries for sequencing and the downstream analysis of raw sequencing data to determine gene expression levels. Much of this work can be carried out with equipment and reagents that are readily available, and the methods can be performed by a majority of laboratory groups. RNA-seq and transcriptomic analyses are set to become even more common in the coming years. The protocols described here will provide a standardized set of methods for applying this powerful technique to the study of N. gonorrhoeae under a variety of conditions.