Perfecthyb hybridization solution

E. coli tRNA mixtures used for the in vitro cleavage assay by CdiA–CT^EC869 were prepared from JM109tr as described (31 (link)). The RNA fraction was deacylated by incubation in 1.8 M Tris–Cl, pH 8.0, for 2 h at 37°C and separated by Hi-Load 16/10 Q-Sepharose HP chromatography, and then the tRNA fractions were pooled and ethanol-precipitated. RNAs with or without CdiA–CT^EC869 treatment were separated on a 10% (w/v) polyacrylamide gel containing 7 M urea and then transferred to a Hybond-N + membrane (GE Healthcare, Japan) by a Trans-Blot SD semi-dry cell (Bio-Rad, Japan). Hybridization was carried out overnight at 60°C in PerfectHyb Hybridization Solution (Toyobo, Japan). The membrane was washed three times with buffer containing 2 × standard saline citrate (SSC) and 0.1% SDS for 5 min and then washed twice with buffer containing 0.1 × SSC and 0.1% (w/v) SDS for 10 min at 60°C. The oligonucleotide sequences used as specific probes for tRNAs are listed in Supplementary Table S2.

A denaturing agarose gel was prepared at a final concentration of 1% (w/v) Agarose ME (Nacalai Tesque), 1× MESA (Dojindo), 2% formaldehyde (Nacalai Tesque), and 0.5 μg/ml ethidium bromide. A total of 250 ng of each RNA sample was mixed with an equal volume of Gel Loading Buffer II (Ambion) and incubated at 65°C for 15 minutes, followed by quick cooling on ice, and then electrophoresed in 1× MESA. The gel was transferred onto a nylon membrane Hybond-N (GE Healthcare) by a capillary blotting method using 10× SSC Buffer (Nacalai Tesque). After UV cross-linking, the membrane was pre-incubated in PerfectHyb Hybridization Solution (TOYOBO) at 65°C for 20 minutes. An RNA probe was added to the solution and incubated at 65°C overnight. The membrane was washed with 2× SSC (+0.1% SDS) and 0.2× SSC (+0.1% SDS) at 65°C for 15 minutes, and then irradiated onto Storage Phosphor Screen BAS-IP (GE Healthcare). Images were scanned using BAS-5000 Image Analyzer (Fujifilm). In order to prepare RNA probes (N(-) and Le(-)), NDV cDNA was subjected to PCR using primer sets including the T7 RNA polymerase promoter sequence (Table in S1 Table). PCR products were in vitro transcribed into [α-³²P]-CTP-radiolabeled RNA probes by Riboprobe System-T7 (Promega). Unincorporated nucleotides within the samples were removed by NucAway Spin Columns (Ambion).