Rq1 rnase free dnase

Virion encapsidated RNA was extracted from viruses (SINV-nLuc) were purified over a 27% sucrose cushion using TRiZOL reagent (Sigma Aldrich) using manufacturer’s protocol. Post extraction, RNAs were DNase (RQ1 RNase-free DNase, NEB) treated using manufacturer’s protocol to remove cellular contaminants and viral RNA copies were quantified via quantitative RT-PCR using primers probing for SINV nsP1 and E1 genomic regions (Supplementary S1 Table) and a standard curve comprised of linearized SINV infectious clone containing the full-length viral genome. To determine infectivity or replication kinetics of Sindbis virion RNA, equal copies of virion isolated RNA (10⁵ copies), quantified using qRT-PCR, were transfected into BHK-21 cells in serum-free Opti-MEM (Gibco). To maximize production of infectious units, equal mass (1 μg) of virion (SINV-nLuc) isolated RNA derived from JW18 fly cells was transfected into BHK-21 cells. Transfection was carried out for 6 hours before the transfection inoculum was removed and overlay was applied. Cells were fixed 48 hours post transfection using 10% (v/v) formaldehyde and stained with crystal violet to visualize plaques.

Split Broc RNA strands were assembled with Coli DNA strands (and vice-versa) by mixing in an equimolar ratio in double-deionized water. The samples were heated to 95 °C for 2 min, snap-cooled to 4 °C for 2 min, and assembly buffer (89 mM tris-borate (pH 8.2), 2 mM MgCl₂, 50 mM KCl) was added, followed by 20 min of incubation at room temperature. Each hybrid split was then added with its opposite hybrid split (e.g., dBroc + rColi was added with rBroc + dColi) in an equimolar ratio at a final concentration of 1 µM with 2/15 volume of RQ1 RNase-free DNase (New England BioLabs). This was incubated at 37 °C for 30 min, then 2/5 of the volume were added to RNase ONE™ (Promega, Madison, WI, USA) at a volume equal to the RQ1 RNase-free DNase added previously. This was incubated at 37 °C for 30 min. Samples were visualized on an 8% non-denaturing native-PAGE (37.5:1) in the presence of 89 mM tris-borate (pH 8.2) and 2 mM MgCl₂ run for 20 min at 4 °C and 300 V.

DNA strands were purchased from Integrated DNA Technologies (Coralville, IA, USA) (Sequences available in Supporting Information) and PCR-amplified using MyTaq™ Mix from Bioline (London, UK). PCR products containing T7 RNA polymerase promoters were purified using the DNA Clean and Concentrator™ kit from Zymo Research (Irvine, CA, USA). RNAs were produced by in vitro run-off transcription with T7 RNA polymerase (80 mM HEPES-KOH (pH 7.5), 2.5 mM spermidine, 50 mM DTT, 25 mM MgCl₂, 5 mM each rNTP). After 3.5 h at 37 °C, the reaction was incubated with RQ1 RNase-free DNase (New England BioLabs, Ipswich, MA, USA) prior to purification using denaturing 8 M urea polyacrylamide gel electrophoresis (PAGE, 15%). RNA bands were visualized under UV (short wavelength), cut, and eluted in crush and soak buffer (300 mM NaCl, 89 mM tris-borate (pH 8.2), 2 mM EDTA) overnight. Precipitation of the RNA was done in 2.5 volumes of 100% ethanol for 3 h at −20 °C. Samples were rinsed with 90% ethanol, vacuum dried, and dissolved in double-deionized water (17.8 MΩ·cm).