DNA oligonucleotides were purchased with standard desalting purification (Thermo Fisher Scientific). Short RNA oligonucleotides under 20-nt and construct 12 were synthesized on a MerMade synthesizer (BioAotomation) using standard phosphoramidite chemistry and deprotected as previously described (16 (link)).
Long RNA oligonucleotides were transcribed in vitro from synthetic ssDNA templates (Thermo Fisher Scientific). Equimolar amounts of ssDNA template and a universal T7 promoter sequence were annealed. One microgram annealed template was then used in a 100 μl transcription reaction containing 5 mM each rNTP, 22 mM magnesium chloride, 40 mM Tris–HCl pH 8.0, 2 mM spermidine, 10 mM DTT, 0.01% Triton X-100, 40 units RNase inhibitor (New England Biolabs) and 5 μl T7 RNA polymerase. Transcription reactions were incubated at 37°C for 2 h.
All RNA was gel purified by denaturing gel electrophoresis. Appropriate bands were excised and RNA was extracted by crush soak in 10 mM MOPS pH 6.0, 300 mM sodium chloride, and 1 mM disodium-EDTA. After extraction, RNA was ethanol precipitated, washed once with 70% ethanol and resuspended in water.
Long RNA oligonucleotides were transcribed in vitro from synthetic ssDNA templates (Thermo Fisher Scientific). Equimolar amounts of ssDNA template and a universal T7 promoter sequence were annealed. One microgram annealed template was then used in a 100 μl transcription reaction containing 5 mM each rNTP, 22 mM magnesium chloride, 40 mM Tris–HCl pH 8.0, 2 mM spermidine, 10 mM DTT, 0.01% Triton X-100, 40 units RNase inhibitor (New England Biolabs) and 5 μl T7 RNA polymerase. Transcription reactions were incubated at 37°C for 2 h.
All RNA was gel purified by denaturing gel electrophoresis. Appropriate bands were excised and RNA was extracted by crush soak in 10 mM MOPS pH 6.0, 300 mM sodium chloride, and 1 mM disodium-EDTA. After extraction, RNA was ethanol precipitated, washed once with 70% ethanol and resuspended in water.
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