RNA extraction and RNA-sequencing method for transcriptomic analysis of <i>Mycobacterium tuberculosis</i>

RNA-sequencing (RNA-seq) technologies have advanced exponentially in recent years, however, the application of RNA-seq to <i>Mycobacterium tuberculosis</i> remains limited. We present a wet-lab and computational protocol for RNA-seq based transcriptomics that was tested on 12 replicates each of 11 clinical isolates of <i>M. tuberculosis</i> (<i>n</i> = 132) grown <i>in vitro</i> with and without pyrazinamide exposure. This RNA extraction method uses low-volume cultures, mechanical lysis, TRIzol^™ phase separation, and column-based purification to produce high yields of pure, intact RNA followed by rRNA depletion and cDNA library preparation. The detection of unique transcripts was optimized at a sequencing depth of 15 million reads. This method detected differential RNA expression in experimental sets with and without pyrazinamide exposure, demonstrating that the method is suitable for RNA-seq applications. We propose a complete wet-lab and computational protocol for RNA-sequencing of <i>Mycobacterium tuberculosis</i> including RNA extraction, rRNA depletion, cDNA library preparation, high-throughput sequencing, and differential expression analysis. This research contributes to the literature by providing a start-to-finish methodology for RNA-sequencing of <i>M. tuberculosis</i>. We describe a complete wet-lab and computational workflow for RNA-sequencing based transcriptomics of <i>M. tuberculosis</i> grown as low-volume cultures <i>in vitro</i>, including: (1) RNA extraction, (2) rRNA depletion, (3) cDNA library preparation, (4) high-throughput sequencing, and (5) differential expression analysis. Created with Biorender.com