RNA Stability Determination Protocol

To determine RNA stabilities, cultures were grown in BHI to exponential phase (OD₆₂₀ ≈ 0.15) as described above, and a culture sample (0 h after the end of log-phase growth [T₀]) was collected. Rifampin was added to inhibit transcription, and additional samples were collected 5, 10, 20, and 30 min after rifampin addition. All samples were subjected to hot phenol lysis as described previously (80 (link)). Briefly, 700 μl of sample was added to a mixture containing 800 μl of acid phenol–chloroform-isoamyl alcohol, pH 4.3 (Fisher Scientific), and 100 μl of lysis buffer (320 mM sodium acetate [pH 4.6], 8% [wt/vol] SDS, and 16 mM EDTA) equilibrated to 65°C. Samples were mixed at 65°C for 5 min and centrifuged for 30 min at 4°C to separate phases. The upper aqueous phase was extracted a second time with an equal volume of neutral phenol–chloroform-isoamyl alcohol, pH 6.7 (Fisher Scientific). RNA was ethanol precipitated and resuspended in DEPC-treated water. RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific).