Transcriptomic Analysis of M. smegmatis sRNA

Total RNA was isolated as previously described (25 (link)). Briefly, M. smegmatis strains carrying the inducible expression vector of 6C sRNA or the empty vector were grown to OD₆₀₀ of ∼0.6. ATc (100 ng/ml) was then added to induce the expression of the sRNA. RNA was extracted at 1- and 3- h after induction using the Qiagen RNeasy Mini kit and treated with the Turbo DNase (Life). After removal of the ribosomal RNA using the RiboZero (Bacteria) kit from Illumina, the purified RNA was used to construct cDNA library according to the TruSeq Stranded RNA LT Guide from Illumina. High-throughput sequencing was carried out on an Illumina HiSeq 2500 system according to the manufacturer's instructions and 150-bp paired-end reads were obtained. The raw reads were filtered by Seqtk and then mapped to the M. smegmatis mc²-155 strain reference sequence (GenBank NC_008596) using Bowtie2 (version: 2–2.0.5) (26 (link)). Counting of reads per gene was performed using HTSeq followed by TMM (trimmed mean of M-values) normalization (27 (link),28 (link)). Differentially expressed genes were defined as those with a false discovery rate Q < 0.05 and fold-change ≥2 using the edgeR software (29 (link)). For each strain, RNA-seq experiment was done twice, one at each time point (1 and 3 h) after Atc induction.