Typhoon fla 7000 phosphorimager

HeLa nuclei treatments, isolations, and nuclear walk-on reactions were done as previously described with slight modification to the walk-on procedure (37 ). In short, 1–3 × 10⁵ nuclei were incubated in 20 mM HEPES pH 7.6, 5 mM Mg(Ac)₂, 5 mM DTT, 100 mM K(Ac), and 0.25 U/μl SUPERase-In with and without both 1.33 μg/ml α-amanitin (Sigma A2263) and approximately 2.5 μg of purified DFF40 at 37°C for 10 min in 24 μl. Immediately after digestion, the solution was raised to 30 μl of 0.5% Sarkosyl, 150 mM K(Ac), and 0.0833 μM α-³²P-CTP to allow for radio-label incorporation for 3 min. Reactions were chased with 500 μM of cold ATP, UTP, GTP, and CTP for 10 minutes. Due to the dramatic increase in viscosity due to the release of DNA from chromatin with Sarkosyl, chasing was performed by tripling the volume to 90 μl and down-up pipetting with a cut pipette tip. Elongation was stopped with stop solution containing 20 mM EDTA, 0.1M Tris, 1% Sarkosyl and 200 μg/ml Torula Yeast RNA to a final volume of 120 μl. Transcripts were isolated by Trizol LS (Ambion 10296028), precipitated by the addition of three volumes of 95% ethanol and 500 mM NH₄(Ac), washed with 70% ethanol, and analyzed using 6% urea–PAGE. Total RNA was visualized by ethidium bromide staining and radiolabeled RNAs were visualized with a Fujifilm Typhoon FLA-7000 phosphorimager.

For the standard polymerase assay, 0.9 μM of the SFTSV L protein was incubated sequentially with 1.8 μM of single-stranded 5′ promoter RNA and 1.8 μM of single-stranded 3′ promoter RNA in assay buffer (100 mM HEPES, 50 mM KCl, 2.5 mM MgCl₂ and 2 mM DTT, pH 7) on ice for 20 min (Supplementary Table S1). For primer-dependent reactions, 18 μM of the respective primer was added (making for a 10-fold molar excess to promoter RNA) to SFTSV L protein bound to promoter RNA and the mix incubated for an additional 20 min on ice. The polymerase reaction was started by the addition of NTPs (0.2 mM ATP/CTP/UTP and 0.1 mM GTP spiked with 166 nM, 5 μCi[α]³²P-GTP, unless stated otherwise) in a final reaction volume of 10 μl. After incubation at 30°C for 2 h, the reaction was stopped by adding an equivalent volume of RNA loading buffer (98% formamide, 18 mM EDTA, 0.025 mM SDS, xylene cyanol and bromophenol blue) and heating the samples at 95°C for 5 min. Products were then separated by denaturing gel electrophoresis using 25% polyacrylamide 7 M urea gels and 0.5-fold Tris–borate–EDTA running buffer. Signals were visualized by phosphor screen autoradiography using a Typhoon FLA-7000 phosphorimager (Fujifilm) operated with the FLA-7000 software.