Superscript 3 first strand synthesis system with random hexamers

Overnight stationary cells were inoculated into 3 ml LB + Sm medium, grown at 37°C until mid-late exponential phase (OD 600 0.5–0.8), harvested and total RNA extracted with TRIzol reagent (Life Technologies). RNA was treated with Turbo DNase I for 30 min (Life Technologies) and subjected to qRT-PCR as previously described [71 (link)]. Briefly, 1 μg total RNA was used for the reverse transcription reaction with Superscript III first strand synthesis system with random hexamers (Life Technologies). The synthesized cDNA was subjected to real time-PCR amplification using the Fast SYBR Green Master Mix kit (Life Technologies) on the StepOnePlus platform (Life Technologies) using primers shown in S6 Table. The amplification data was analyzed by ΔΔCT method utilizing rpoC mRNA as internal control.

Cells from overnight (stationary-phase) cultures were inoculated in triplicate into 5 ml LB or M9 and grown at 37°C until exponential phase (OD₆₀₀ of ∼0.3) or stationary phase. Total RNA was extracted from harvested cells with TRIzol reagent (Life Technologies). RNA was treated with Turbo DNase I for 30 min (Life Technologies) and subjected to quantitative reverse transcriptase PCR (qRT-PCR) as previously described (38 (link)). Briefly, 1 μg total RNA was used for the reverse transcription reaction with Superscript III first strand synthesis system with random hexamers (Life Technologies). Real-time PCR amplification of the synthesized cDNA was conducted using the Fast SYBR green Master Mix kit (Life Technologies). Each of the three biological replicates was analyzed in technical triplicate on the StepOnePlus platform (Life Technologies) using primers shown in Table S3. The data were analyzed by the ΔΔC_T method using rpoC mRNA as an internal control. Log₂ fold change was calculated from the ΔΔC_T results.