Preparation of RNA, agarose gel electrophoresis, and northern blotting

Samples (5 mL) were taken from 75 mL cultures and quenched with 1 mL of 5% phenol (in ethanol) at room temperature. Zero min time points were taken immediately after addition of rifampicin (150 µg/mL). After centrifugation (3000 g, 20 min, 4°C), supernatants were decanted, residual liquid was removed with a Kimwipe, and cell pellets were stored at −20°C. Total RNA was extracted using a Direct-zol RNA MiniPrep Plus kit (Zymo Research) as described (Hadjeras et al. 2019) except the DNase step was omitted. Briefly, cells were lysed under strongly denaturing conditions in TRIzol. RNA was column purified and eluted in 50 µL of water. RNA amounts were quantitated by UV absorption using a Nano Drop spectrophotometer.

RNA was fractionated on mini format 2.4% agarose gels in 1× TBE at 100 V for 40 min. Samples were either mixed directly with a 2× formamide loading buffer or dried in a SpeedVac and suspended in 1× formamide loading buffer. Before loading, samples were heated in a ThermoMixer (50°C, 720 rpm, 5 min). Gels were stained with SYBR Safe and imaged with a GelDoc system. Northern blotting was performed as described (Hadjeras et al. 2019).

rRNA and tRNA species were quantified using Image Lab software. In Figures 2, ​,4,4, 6 and 7, nominal amounts of RNA were determined using known quantities of the 750 and 1500 bp double-stranded DNA size markers as standards (1 kb ladder, ThermoScientific). In these figures, the quantification is presented as a log₂ plot to better visualize RNA levels that vary as much as 50-fold in some measurements.