Transcriptomic dataset of Mycolicibacterium smegmatis exposed to an imidazo[1,2-b][1,2,4,5]tetrazine

Deciphering the mechanism of action of novel anti-tuberculosis compounds is a key step in the drug development process. We have previously described a number of imidazo[1,2-b][1,2,4,5]tetrazines with a promising activity on Mycobacterium tuberculosis[1]. These compounds had predicted activity as serine‑threonine protein kinase inhibitors, however spontaneous drug resistant Mycolicibacterium smegmatis mc2155 (formerly Mycobacterium smegmatis) revealed only the mycobacterial mechanism of resistance to imidazo[1,2-b][1,2,4,5]tetrazines: mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis, thus providing resistance to imidazo[1,2-b][1,2,4,5]tetrazines via enhanced efflux [2]. Here we report the RNA sequencing data of M. smegmatis mc2 155 culture treated with one of the imidazo[1,2-b][1,2,4,5]tetrazines for 1.5 h and the untreated culture as a control. The mapped reads showed that a total of 1386 genes are differentially expressed in this experiment. A further analysis of these data can shed light of the mechanism of action of imidazo[1,2-b][1,2,4,5]tetrazines. The data generated by RNA-seq (raw reads) have been deposited to NCBI sequence read archive (SRA) and have been assigned a BioProject accession number PRJNA615922.

RNA sequencing Transcriptome imidazo [1,2-b ] [1,2,4,5]tetrazines Mycolicibacterium smegmatis Drug development Tuberculosis DEGs a b s t r a c t Deciphering the mechanism of action of novel antituberculosis compounds is a key step in the drug development process. We have previously described a number of imidazo [1,2-b ] [1,2,4,5]tetrazines with a promising activity on Mycobacterium tuberculosis [1] . These compounds had predicted activity as serine-threonine protein kinase inhibitors, however spontaneous drug resistant Mycolicibacterium smegmatis mc 2 155 (formerly Mycobacterium smegmatis ) revealed only the mycobacterial mechanism of resistance to imidazo [1,2-b ] [1,2,4,5]tetrazines: mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis , thus providing resistance to imidazo [1,2-b ] [1,2,4,5]tetrazines via enhanced efflux [2] . Here we report the RNA sequencing data of M. smegmatis mc 2 155 culture treated with one of the imidazo[1,2b ][1,2,4,5]tetrazines for 1.5 h and the untreated culture as a control. The mapped reads showed that a total of 1386 genes are differentially expressed in this experiment. A further analysis of these data can shed light of the mechanism of action of imidazo [1,2-b ] [1,2,4,5]tetrazines. The data generated by RNA-seq (raw reads) have been deposited to NCBI sequence read archive (SRA) and have been assigned a Bio-Project accession number PRJNA615922.
© 2020 The Author(s

Data description
The dataset presented in this article represents raw RNA-seq reads from samples of Mycolicibacterium smegmatis mc 2 155 treated with compound 3a ( Fig. 1 ) Table).

Bacterial strains and growth conditions
M. smegmatis mc 2 155 strain was used in this work. M. smegmatis cultures were grown in Middlebrook 7H9 medium (Difco Becton Dickinson, USA) supplemented with 0.5% (v/v) glycerol and 0.05% (v/v) Tween 80 at 37 °C and 250 rpm.

Experimental design
M. smegmatis cultures were grown overnight in Middlebrook 7H9 broth to mid-log phase (OD600 = 1.0-1.2) and then compound 3a dissolved in DMSO was added to the medium to a final concentration of 256 μg/ml (4 × minimal inhibitory concentration [1] ) for 1.5 h. The same amount of DMSO was added to the control samples. Afterwards cells were washed twice with fresh ice-cold Middlebrook 7H9 broth and total RNA was isolated. In total, 6 RNA samples were obtained -3 biological replicates in the control and experimental conditions.

RNA extraction and sequencing
Cells from 10 mL culture were harvested by centrifugation for 10 min at 30 0 0 × g and 4 °C, washed twice by 10 ml of fresh Middlebrook 7H9 broth and once by 1 ml of RNAprotect Bac-teria Reagent (Qiagen, USA). Total RNA was extracted as described by Rustad et al. [3] , with some modifications. In brief: M. smegmatis cells were homogenized in ExtractRNA reagent (Evrogen, Russia), followed by phenol (pH = 4.5)-chloroform/isoamyl alcohol (25:24:1) purification and precipitation with isopropanol (2:1, v/v). Remaining genomic DNA was removed by DNAse I, Amplification grade (Invitrogen, USA). Total RNA (1 μg) was used for library preparation. Ribosomal RNA was removed from the total RNA using the RiboMinus Transcriptome Isolation Kit, bacteria (Thermo Fisher Scientific) and libraries were prepared using the NEBNext R Ultra II Directional RNA Library Prep Kit (NEB), according to the manufacturer's protocol. Libraries were subsequently quantified by Quant-iT DNA Assay Kit, High Sensitivity (Thermo Fisher Scientific). Finally, equimolar quantities of all libraries (12 pM) were sequenced by a high throughput run on the Illumina HiSeq using 2 × 100 bp paired-end reads and a 5% Phix spike-in control.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.