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 Mycobacterium tuberculosis 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 M. tuberculosis ( n = 132) grown in vitro 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.