Dnase removal reagent

Cells were grown at 30 °C in LB medium overnight and subcultured at a 1:100 ratio into fresh LB medium. After 6 h of growth at 37 °C in the presence or absence of TCS activator as specified, aliquots of 1 ml of cell culture were harvested by centrifugation. Total RNA was extracted using a RNeasy Mini Kit following the manufacturer’s instructions (Qiagen Sciences, Germantown, MD), and treated with Turbo-DNA-free DNase (Ambion). The DNase was removed using DNase removal reagents (Ambion). RNA samples were quantified using a NanoDrop spectrophotometer. Total RNA (200 ng) from each sample was subjected to cDNA synthesis using high-capacity cDNA reverse transcription kits (Applied Biosystems, Foster City, CA). Quantitative PCR (qPCR) was then conducted using iQ SYBR green supermix (Bio-Rad) on a CFX96 qPCR cycler (Bio-Rad). The B. anthracis housekeeping gene 16 S rRNA was used as an internal control.

Total RNA was extracted using a RNeasy Mini Kit following the manufacturer’s instructions (Qiagen Sciences, Germantown, MD), and treated with Turbo-DNA-free DNase (Ambion). The DNase was removed using DNase removal reagents (Ambion). RNA samples were quantified using a NanoDrop spectrophotometer. RNA (15 μg) was mixed with loading buffer (formamide, bromophenol blue, xylene cyanol, EDTA), denatured at 90 °C for 2 min, loaded on a 1% bleach agarose gel, and run at 100 W for ~2 h. RNA was transferred onto nylon membrane in 20× SSC buffer at room temperature overnight followed by cross-linking two times at 254 nm. The membrane was incubated in DIG easy hybridization buffer at 42 °C for 1 h. Four antisense-oligo probes against hitPRS (Supplementary Table 2) were pooled together and labeled at 5’-ends with [γ-³²P]-ATP using T4 polynucleotide kinase. After labeling, G10 columns (NucAway spin columns, Invitrogen) were used to remove the unincorporated (γ-³²P) ATP. The labeled probes were added to the hybridization buffer and incubated at 42 °C overnight. One labeled probe against 16 S rRNA (Supplementary Table 2) served as a loading control. The membrane was washed 3× in wash buffer (0.5× SSC, 0.1%SDS) followed by exposure to a phosphorimager screen overnight, and the radioactive signals were detected using a phosphor image analyzer (Typhoon FLA 7000).

Cells were grown at 30°C in LB medium overnight and subcultured at a 1:100 ratio into fresh LB medium. After 6 h of growth at 37°C in the presence or absence of 20 μM ‘205, aliquots of 4 ml of cell culture were harvested by centrifugation. Total RNA was extracted using RNeasy Mini Kit following the manufacturer’s instructions (Qiagen Sciences, Germantown, MD), and treated with Turbo-DNA free DNase (Ambion). The DNase was removed using DNase removal reagents (Ambion). RNA samples were quantified using a NanoDrop spectrophotometer. Two hundred nanograms of total RNA from each sample was subjected to cDNA synthesis using high-capacity cDNA reverse transcription kits (Applied Biosystems, Foster City, CA). Quantitative PCR (qPCR) was then conducted using iQ SYBR green supermix (Bio-Rad) on a CFX96 qPCR cycler (Bio-Rad). The B. anthracis housekeeping gene 16S rRNA was used as an internal control.