Gene Expression Analysis of Persisters

For the analysis of comC gene expression in stationary-phase persisters, overnight cultures were diluted (1:100) in fresh THYE broth and incubated at 37 °C for ~10 h. Stationary-phase cultures were then exposed to 20 µg/mL of ofloxacin for 24 h at 37 °C. Cells were harvested via centrifugation and washed with sterile PBS before being processed for total RNA extraction using a RiboPure-Bacteria purification kit (Thermo Fisher Scientific Inc.), as described previously [19 (link)]. Quantitative reverse-transcription PCR (RT-qPCR) analysis was performed using Forget-Me-Not EvaGreen qPCR Master Mix (Biotium, Fremont, CA, USA) and a CFX96 real-time PCR detection system (Bio-Rad, Mississauga, ON, Canada). Data analysis was performed using relative quantification normalized against unit mass. For the analysis of relE40 gene expression, overnight cultures were diluted (1:100) in fresh THYE broth and incubated until mid-log phase. Cultures were then exposed for 2 h at 37 °C to the following DNA damage conditions: quinolone (2 µg/mL), mitomycin C (0.5 µg/mL), hydrogen peroxide (0.5 mM), and pH 5.0. Cells were then harvested via centrifugation and washed with PBS before being processed for total RNA extraction [19 (link)]. RT-qPCR analysis was performed as described above. Primers used for RT-qPCR are listed in Table S1.