Vaccinia capping system kit

RNAs used in RNA chromatography experiments were transcribed using the MEGAscript Kit (Invitrogen AM1333) as per manufacturer’s instructions. 7.5 mM ATP/CTP/GTP, 6.75 mM UTP and 0.75 mM biotinylated UTP (Biotin-16-UTP, Lucigen BU6105H) were used. For luciferase reporter mRNAs, m⁷G-cap was added using the Vaccinia capping system kit (M208S NEB) according to the manufacturer’s instructions. To generate the DNA templates to synthetize the luciferase reporter mRNAs, oligonucleotides CDC45, FAS and GADD45α were amplified by PCR using the following primers. For CDC45, PCR products were used as template for in vitro transcription. For GADD45α and FAS, PCR products were cloned in the pSC-B-amp/kan plasmid from the Strataclone Blunt PCR Cloning Kit, and then digested by, respectively, BamHI and XhoI (restriction sites for transcription run-off) and purified. All oligonucleotide sequences are available in the following table (bold: T7 promoter). For GADD45α, T7 sequence used was in pSC-B-amp/kan plasmid.

For IVT, the different plasmids were linearized with the type IIS restriction enzyme BspQI and purified with the Monarch PCR & Cleanup Kit (NEB). Capped or uncapped mRNAs were produced with the T7 ULTRA mMESSAGE mMACHINE Kit (Ambion, Life Technologies, France) or the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB), respectively. The reactions were done according to the manufacturer’s recommendations. Then, the IVT mRNAs were precipitated with LiCl and their integrity evaluated by agarose gel electrophoresis. The enzymatic mRNA capping was performed with the Vaccinia Capping System Kit (NEB) according to the manufacturer’s recommendations. Vaccinia virus-capped mRNAs were precipitated by adding 1 volume of isopropanol 100% and 0.1 volume of 3 M sodium acetate, pH 5.5. After 30 min at −20°C, the RNA solution was centrifuged (19,000 × g, 4°C, 45 min) and the pellet resuspended in nuclease-free water. The enzymatic capping efficiency was validated by fluorescence microscopy after mRNA transfection into HeLa cells.