Northern Blot Analysis of sRNAs

Detection of sRNAs in Northern blot experiments was carried out as described [5 (link), 35 (link)]. sRNAs (25–35 μg) were separated by electrophoresis on 15% TBE-Urea gels (Invitrogen), electrotransferred to Hybond N1 filters at 400 mA for 1 h in 0.5X TBE, and cross-linked by ultraviolet irradiation (2 pulses at 1.2x10⁵ μJ per cm²). Ultrasensitive hybridization buffer UltraHyb (Ambion) was used for prehybridization and hybridization. Antisense-specific riboprobes were prepared by in vitro transcription (MAXIscript transcription kit; Ambion) and labeled with [α-32P] dUTP following supplier-recommended protocols. Riboprobes were treated as described previously [7 (link)] to result in an average size of 50 nucleotides (nt). An antisense-specific probe for the fkbA gene was amplified from genomic DNA with primers JOHE23654 and JOHE23559 and in vitro transcribed from the T7 promotor contained within the JOHE23654 primer sequence (S2 Table). This in vitro transcribed probe was also used to detect antisense fkbA mRNA in the different strains by Northern blot (S10 Fig). The carB antisense-specific riboprobe was obtained from the in vitro transcription of linearized plasmid pMAT652 [24 (link)]. JOHE37682 and JOHE37683 were used to amplify the 5S rRNA from genomic DNA and in vitro transcribed from the T7 promotor contained in both primer sequences (S2 Table).