Tbe buffer

A 3-fold molar excess of monovalent streptavidin was added to the fluorophore-tagged desthiobiotinylated ds39 RNA (25 nM) at 4°C in Buffer A for 30 min. The EMSA reactions used a range of RIG-I or LGP2 concentrations (as indicated in the legend) from 0.75- to 12-fold molar excess relative to RNA. The samples were loaded onto a 4–20% gradient native polyacrylamide gel in TBE buffer (Invitrogen).

The EMSA reactions were carried out as above utilizing equal or double molar concentrations (20, 40 nM) of RIG-I or LGP2 and the fluorophore-tagged dsRNA (20 nM) and Buffer A supplemented with 100 mM NaCl. Subsequently, the reactions were loaded onto a 20% native polyacrylamide gel in TBE buffer (Invitrogen).

RNase P cleavage was performed by mixing 300 nm MRPP1–MRPP2, 150 nm MRPP3, 10 units of RNase inhibitors (RNasin from Promega), and 400 nmin vitro transcribed pre-(mt) tRNA^Ile in a buffer of 30 mm Tris-HCl, pH 8, 40 mm NaCl, 4.5 mm MgCl₂, and 2 mm DTT to a total reaction volume of 8.25 μl. The reaction was performed at room temperature and stopped at set times by transferring 1 μl of the reaction mixture into 5 μl of 500 mm EDTA and heating to 95 °C. This sample was analyzed by denaturing PAGE on a 6% TBE-urea gel (Invitrogen) run in TBE buffer (Invitrogen). The gel was stained for 30 min with SYBR Gold (Invitrogen) diluted 1000-fold in TBE buffer from the manufacturer's stock and imaged using the Bio-Rad ChemiDoc imaging system.

A Bacillus species-specific chromosomal region was amplified with a PCR assay based on Yamada et al. [34] (link). Bacterial primers were used to amplify a fragment of the gyrB gene of B. cereus (BC1 and BC2r, 365 bp), B. anthracis (BA1 and BA2r, 245 bp) and B. thuringiensis (BT1 and BT2r, 368 bp) (Table 1). Oligonucleotide primers BC1, BC2r, BA1 and BA2r were obtained from Eurogentec S.A. and BT1 and BT2r from Biolegio. Template DNA was prepared as described above using whole bacterial cells or DNA solution in duplicate (four-fold, in total). All PCR reactions were performed in a total volume of 25 µl containing 1 or 5 µl of template DNA, 0.25 U Super TAQ HC (HT Biotechnology Ltd.), PCR Buffer (HT Biotechnology Ltd.), 200 µM of each dNTP (Fermentas Int. Inc.), 200 nM of each primer and 1 mM MgCl₂. Amplification was carried out on a PTC-225 DNA Engine Tetrad (MJ Research Inc., Massachusetts, USA) as described previously [34] (link). To confirm amplification of PCR products of the proper sizes, electrophoresis was performed through an ethidium bromide stained 1.0% (w/v) agarose gel (Eurogentec S.A.) in TBE buffer (Invitrogen Ltd.).

Primers, from Eurofins Genomics (detailed sequence information in Table S1), were tested for hybrids and hairpin structures using the Integrated DNA Technologies design tools (http://eu.idtdna.com/pages/scitools). The HCR reaction contained 0.1 μM of each primer (NP1/NP2/NP3) and initiator, 50 mM of Na₂HPO₄, and 0.5 M of NaCl. All the primers were heated upto 95 °C for 5 min and cooled down to room temperature naturally^{6 (link)}, before adding the initiator. The HCR reaction was performed at room temperature overnight. The products of the reaction were analyzed in 3% agarose gels with SYBR gold (Invitrogen) in 1 × TBE buffer (Invitrogen). Gels were run at 75 V for 120 min and visualized under with SYGENE PXi. Anonymized EDTA Blood was obtained commercially (Cambridge Bioscience Ltd) from healthy volunteers and stored at 4 °C, until used and discarded after 8 days in storage.