Riboruler high range ladder

Total RNA (15 μg) was separated on a 1% agarose gel (1X MOPS (20 mM MOPS free acid, 5 mM sodium acetate, 1 mM EDTA, pH 7.0), 6.6% formaldehyde), in 1X MOPS buffer containing 0.7% formaldehyde. The RNAs were transferred onto a Nylon Hybond N+ membrane (GE Healthcare). The transfer was carried out on a capillary system overnight at room temperature in 20X SSC buffer. The cross-linking of RNAs on the 6X SSC-rinsed membrane was performed with UV (2X autocrosslinking, UV Stratalinker 1800). The oligonucleotides primers (Supplementary Table I) were labelled with gamma-³²P ATP (Hartmann analytics) using the T4 Polynucleotide Kinase (T4-PNK, Fermentas) and purified over G-25 columns (GE Healthcare) as previously described [33]. The denatured primers were incubated with the membranes in hybridization buffer (Rapid-hyb buffer, GE Healthcare) overnight at 50°C. The membranes were washed with 1X SSC + 0.1% SDS for 20 min at 50°C and subsequently with 0.5X SSC + 0.1% SDS. The radioactive signal was visualized after 2 or 3 days of exposure using a phosphorimager (Typhoon Fla 9000, Fujifilm). 16S rRNA was used as a loading control. The approximate sizes of RNA transcripts were estimated using the RiboRuler High Range Ladder (Thermo Scientific). Each experiment was performed at least in independent triplicates.

Total RNA was prepared by acid-phenol extraction after mechanical cell disruption as described previously (Nicolas et al., 2012 (link)). The quality of the RNA was assessed by means of an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States) according to the manufacturer’s instructions. Northern blot analysis was performed as described before (Homuth et al., 1997 (link)). Transcript sizes were determined using the RiboRuler High Range Ladder (Thermo Fisher Scientific, Waltham, MA, United States) and the digoxigenin-labeled RNA Molecular Weight Marker II (Roche Holding AG, Basel, Switzerland). The bands of the unlabeled RNA size standard were marked directly on the membrane, thereby becoming visible as negative bands during detection that were then indicated by lines in the northern blot images. Quantification of the 3.5 kb opuB transcript and the 1.2 kb S1290 transcript was performed using Image Studio Lite (version 5.2.5, LI-COR Biosciences, Lincoln, NE, United States).