Megascript t7 rna polymerase kit

In vitro transcription of roX2 and GFP RNA was performed using the MegaScript T7 RNA polymerase kit (Ambion) or T7 polymerase (NEB with Ambion buffers and nucleotides), following the manufacturer's instructions. The DNA templates were prepared by restriction enzyme digestion with XbaI (NEB) in the case of roX2 constructs as described (41 (link)). GFP templates were amplified by PCR (for primers, see Supplementary Table) from an pHSP70-MLE-GFP plasmid (52 (link)). After transcription (4 h, 37°C) and DNase I treatment (30 min, 37°C), RNA products were purified by denaturing PAGE (8 M urea, 5% acrylamide, 1x TBE), phenol:chloroform:isoamylalcohol (Roth) extraction and ethanol precipitation. RNA was dissolved in 50 μl of nuclease-free water (Ambion) and stored at –70°C. Total RNA from female Drosophila Kc cells for EMSA was extracted using the RNeasy Mini Kit (Qiagen).

Messenger RNAs were generated as previously described [19 (link)], using linearized plasmids (pTEV-CPD-PL-A101 and pTEVeGFP-A101) encoding codon-optimized Potorous CPD-photolyase (CPD-PL Ψ-mRNA) and enhanced green fluorescent protein (eGFP Ψ-mRNA). The CPD-photolyase gene from Potorous tridactylus (rat kangaroo) was synthesized by Entelechon (Bad Abbach, Germany). The Megascript T7 RNA polymerase kit (Ambion, Austin, TX) was used for transcription, and UTP was replaced with pseudouridine triphosphate (TriLink, San Diego, CA) [21 (link)]. To remove the template DNA Turbo DNase (Ambion) was added to the reaction mix. Pseudouridine-modified mRNAs were HPLC-purified as described [36 (link)] and provided with cap1 generated by using the m7G capping enzyme and 2′-O-methyltransferase according to the manufacturer (CellScript, Madison, WI). The mRNAs were transcribed to contain 101 nt-long 3’ poly(A) tail. Small aliquots of RNA samples were stored in siliconized tubes at -20°C.