Plasmids carrying 9S and RprA genes were provided by A.J. Carpousis and K. Papenfort, respectively. Forward PCR primers were designed in a way to add promoter sequence recognized by T7 RNA polymerase. PCR products were used as In Vitro Transcription (IVT) templates. IVT was carried out according to standard protocol with addition of 3% DMSO (v/v), followed by DNA template digestion with TURBO DNase (Thermo Fisher). For synthesis of 5′ monophosphorylated RNA, five-fold excess of rAMP or rGMP over rATP or rGTP was used for RprA and 9S respectively to cap the product (Bandyra et al., 2012 (link)). Synthesized RNAs were purified on 4% (9S) or 6% (RprA) polyacrylamide gel containing 7.5 M urea (National Diagnostics). The bands were visualized with UV shadowing and excised, and RNAs were eluted from gel slices by overnight electroelution (100V, EluTrap, Whatman). The ompD RNA was prepared as previously described (Bandyra et al., 2012 (link)).
Primers used for IVT template preparation
| Primer name | Sequence 5′ → 3′ | |
| RprA | For | GTTTTTTTTTTAATACGACTCACTATTACGGTTATAAATCAACACATTG |
| Rev | AAAAAAAAGCCCATCGTAGGAG | |
| 9S | For | GTTTTTAATACGACTCACTATAGAAGCTGTTTTGGCGGATGAGAG |
| Rev | CGAAAGGCCCAGTCTTTCGACTGAGC | |
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