RNA extraction and RNA-sequencing method for transcriptomic analysis of Mycobacterium tuberculosis

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.

We propose a complete wet-lab and computational protocol for RNA-sequencing of Mycobacterium tuberculosis 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 M. tuberculosis.

We describe a complete wet-lab and computational workflow for RNA-sequencing based transcriptomics of M. tuberculosis grown as low-volume cultures in vitro, including: (1) RNA extraction, (2) rRNA depletion, (3) cDNA library preparation, (4) high-throughput sequencing, and (5) differential expression analysis.

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