Computational protocol: Essentiality of mmpL3 and impact of its silencing on Mycobacterium tuberculosis gene expression

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Protocol publication

[…] The mmpL3 conditional mutant TB416 was grown until exponential phase with or without ATc [500 ng/ml] in roller bottles (two biological replicates per condition). As a control for the effect of ATc alone, ATc-treated and untreated parental strain was used (one sample per condition). Total RNA was extracted as previously described, and used for the preparation of strand-specific Illumina libraries, with an additional ribosomal RNA depletion step. Ribosomal transcript depletion was performed on 1 μg of total RNA using the Ribo-Zero rRNA Removal Kit for Gram-Positive Bacteria (Catalog Number MRZGP126; Illumina, San Diego, USA) according to the protocol provided by the supplier. Sequencing libraries were then generated using the resulting ribosomal transcript-depleted RNA and the Illumina TruSeq Stranded mRNA Library Prep Kit reagents (Catalog Number RS-122-2101) according to the protocol provided by the supplier. Sequencing cluster generation was performed with the resulting libraries and the Illumina TruSeq SR Cluster Kit v4 reagents (Catalog Number GD-401-4001) and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit V4 reagents (Catalog Number FC-401-4002) as single-end 100 nt-long reads. Sequencing data were processed using the Illumina Pipeline Software version 1.84. Reads were right-trimmed for the Illumina adapter sequence using Flexbar (https://sourceforge.net/projects/flexbar/) and aligned with Bowtie2. Counting reads over annotated features was done with featureCounts. Annotation was taken from TubercuList release R27. To avoid the influence of transcripts deriving from the original 5′ fragment of mmpL3, a truncated version of mmpL3 was added to the annotation. Differential gene expression (DGE) analysis was done using DESeq2. Raw and processed data were deposited in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) as Series record GSE89830. […]

Pipeline specifications

Software tools Flexbar, Bowtie2, Subread, DESeq2
Databases TubercuList MMpl
Application RNA-seq analysis
Organisms Mycobacterium, Bacteria, Homo sapiens
Diseases Tuberculosis