Synthesis of cDNA and RT-PCR

Exponentially growing bacterial cultures (OD₆₀₀ 0.4–0.6) were pelleted and washed once with 7H9 containing 0.05% Tween-80, then diluted to OD₆₀₀ = 0.5. The cells were treated with different stresses, including 5 mM H₂O₂, 5 mM NO, acidic medium (pH4.5), 1%SDS and were incubated for 2 h. Equal amounts of bacteria of M. bovis strains were collected. Total RNA and cDNA from M. bovis BCG were extracted as previously described (Rustad et al., 2009). Briefly, the collected cultures were harvested by centrifugation. After removal of the supernatant, the cell pellet was resuspended in RNA-easy Isolation Reagent (Vazyme) and disrupted by bead beating with a TissueLyser-II (QIAGEN). Total RNA was precipitated by the addition of isopropanol and collected by centrifugation, the supernatant was discarded, and the mRNA pellet was washed with 75% ethanol. After drying of the pellet, mRNA was dissolved in RNase-free H₂O. The contaminating genomic DNA was digested with gDNA wiper Mix (Vazyme). The cDNA was prepared from purified mRNA with HiScriptII qRT SuperMix II (Vazyme) through reverse transcription. The cDNA levels of target genes were then quantified by quantitative real-time PCR (qRT-PCR) on a CFX96 cycler (BIO RAD) by using AceQ Universal SYBR qPCR Master Mix (Vazyme) as a dye for fluorescence signal detection. All qPCR primers were determined to be >95% efficient, and the cDNA masses tested were experimentally validated to be within the linear dynamic range of the assay. Signals were normalized to those of the housekeeping sigA transcript and quantified with the ΔΔCt method. Error bars are 95% confidence intervals of the three technical replicates.