DNA templates were designed to include the 20-nt T7 RNA polymerase promoter sequence (5’-TTCTAATACGACTCACTATA-3’) followed by the remaining sequence encoding desired RNA. Double-stranded templates were prepared by extension of 60-nt DNA oligomers (IDT, Integrated DNA Technologies) with Phusion DNA polymerase (Finnzymes), using the following thermocycler protocol: denaturation for 30 sec at 98°C, 35 cycles of denaturation for 10 sec at 98°C annealing for 30 sec at 60 to 64°C , extension for 30 sec at 72°C; final extension for 10 min at 72 °C and cooling to 4°C.
DNA samples were purified with AMPure magnetic beads (Agencourt, Beckman Coulter) following manufacturer’s instructions. Sample concentrations were estimated based on UV absorbance at 260 nm measured on Nanodrop 100 or 8000 spectrophotometers. Verification of template length was accomplished by electrophoresis of all samples and 10-bp and 20-bp ladder length standards (Thermo Scientific O’RangeRuler SM1313 & SM1323) in 4% agarose gels (containing 0.5 mg/mL ethidium bromide) and 1x TBE (100 mM Tris, 83 mM boric acid, 1 mM disodium EDTA). All sample manipulations, including following steps, were carried out in 96-well V-shaped polypropylene microplates (Greiner).
DNA samples were purified with AMPure magnetic beads (Agencourt, Beckman Coulter) following manufacturer’s instructions. Sample concentrations were estimated based on UV absorbance at 260 nm measured on Nanodrop 100 or 8000 spectrophotometers. Verification of template length was accomplished by electrophoresis of all samples and 10-bp and 20-bp ladder length standards (Thermo Scientific O’RangeRuler SM1313 & SM1323) in 4% agarose gels (containing 0.5 mg/mL ethidium bromide) and 1x TBE (100 mM Tris, 83 mM boric acid, 1 mM disodium EDTA). All sample manipulations, including following steps, were carried out in 96-well V-shaped polypropylene microplates (Greiner).