Rnase 3

Cells were cytospun onto poly-L-lysine coated slides (cat. #67–762; Thermo Fisher Scientific) and fixed in 3.7% paraformaldehyde (cat. #BM1585; Thermo Fisher Scientific), permeabilized with 1× PBS containing 0.2% Triton X-100 (cat. #MTX15681; Thermo Fisher Scientific) for 5 min at RT, and washed with 1× PBS at RT three times for 5 min each time. Cells were then incubated with 20 μg/ml proteinase K (cat. #P9107; New England Biolabs), in 50 mM Tris-HCl, pH 8.0, and 5 mM CaCl₂ for 1 h at 37°C. For RNase III digestion, the slides were washed twice and incubated in RNase III (cat# AM2290; Invitrogen) at least 30 min after proteinase K treatments. Primary antibodies against dsRNA (9D5 or J2; Table S8) were incubated overnight at 4°C (diluted 1:100 in 1× PBS), washed three times at RT for 5 min each time in PBS before incubation with a rabbit anti-mouse AF488 secondary antibody (cat. #31584; Life Technologies) for 30 min, and counterstained with 1 μg/ml DAPI (cat. #10236276001; Millipore Sigma) in 1× PBS for 3 min at RT. Slides were washed once with 1× PBS containing 0.05% Triton X-100 for 5 min each time at RT, twice with 1× PBS for 5 min each time, and once with distilled H₂O for 1 min. Cells were mounted with Prolong Gold antifade gel mount (cat. #P36930; Thermo Fisher Scientific) and imaged on a Cytation 5 inverted fluorescent microscope (BioTek).

In vitro transcribed +1 dinQ, +44 dinQ, and agrB RNA was 3′ end labeled with T4 RNA Ligase (Ambion) and equimolar amounts of [³²P]pCp (PerkinElmer, NEG019A) according to the protocol. Unincorporated [³²P]pCp was removed by applying the mixture to an RNase-free Sephadex G25 column. Folding of RNA prior to RNase III cleavage was performed as described for chemical probing of RNA; the RNA was heated to 95°C for 1 min and placed on ice before folding at 37°C for 10 min in folding buffer followed by addition of RNase III (Ambion). The reaction was stopped by adding an equal volume of Gel Loading Buffer II (Ambion) before being analyzed on a denaturing 5% polyacrylamide/8 M urea gel, and visualized on Typhoon 9410 (Amersham). An aliquot of the RNase III reaction was left for primer extension analysis.