Transcriptaid t7 high yield kit

The DNA template constructs for IC, ICU, IC_d3.1, IC_d3.2, IC_d3.3, C, CRE + HV, U, I + CU and CU RNA were obtained by amplification using specific primers as previously described^{11 (link),22 (link),24 (link)}.
The template for RNA-100 was generated by endonuclease digestion of the pBS SK(+) plasmid with XbaI. RNA667 was obtained as previously reported^{22 (link)}.
RNA molecules were in vitro synthesised using the TranscriptAid T7 High Yield Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. The quantity of RNA produced was determined by UV spectrophotometry (A260); protein and carbohydrate/phenolic contamination was assessed via the A260/A280 and A260/A230 ratios respectively. The integrity of the transcripts was tested by denaturing agarose gel electrophoresis. Internally radiolabelled RNA molecules were synthesised as previously described^{22 (link)}.

DNA coding for the CRE transcript was obtained by PCR amplification, ensuring the presence of the precise 3′ end. Briefly, T7pC + HV was amplified from plasmid pU3′HCV9181 using the primers T7pHCV-918133 and asHCV9414 (5′-AACAGGATGGCCTATTGGCCTG-3′) to obtain the template for CRE plus the downstream hypervariable region (9181–9414). A shorter amplicon, CRE (9181–9384), was amplified using T7pHCV-9181 and as HCV9384 (AGCTCCCCGTTCATCGGTT) primers. The template for RNA 667 was derived from the pcDNA3 vector (Invitrogen) linearized with DraIII51 . RNA synthesis was performed using the TranscriptAid T7 High Yield Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. The resulting transcript was purified as previously described52 . The RNA concentration was determined by UV spectrophotometry (A₂₆₀) and the degree of protein and carbohydrate/phenolic contamination assessed from the A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios respectively. The integrity of the RNA was confirmed by denaturing agarose-formaldehyde gel electrophoresis.
RNA construct (ICU) containing a luciferase reporter gene flanked by the HCV genomic ends was obtained by amplification of pGLICU plasmid with 5′pT7HCV and 3′HCV primers22 , in vitro transcription and purification as explained above. The generation of a capped RLuc RNA was generated as previously described51 .

Plasmid pcDNA3-pri-let-7e (Addgene #51380; Heo et al., 2008 (link)) linearized by digestion with XbaI (NEB) was used as a template for RNA in vitro transcription in the presence of [α-³²P]-GTP (Perkin Elmer) using TranscriptAid T7 High Yield Kit (Thermo Fisher). The resulting RNA (369bp) was denatured by incubation at 73°C for 3 min in 2X Urea Sample Buffer (Thermo Fisher) and run in a 6% TBE–urea precast polyacrylamide gel (Thermo Fisher). The band corresponding to the expected size of pri-let-7e transcript was excised using autoradiography and, after breaking the gel, RNA was eluted as described above for mature miRNA isolation.
2.5 μg of ³²P-labeled pri-let-7e (1 μM) were incubated for 3 h at 37°C in the presence of recombinant METTL1/WDR4 (300 nM) and S-adenosylmethionine (1 mM) in a methylation buffer (85 mM Tris-HCl pH 8.0, 1.4 mM DTT, 0.07 mM EDTA, 1 mM spermidine). Methylated pri-let-7e was isolated by immunoprecipitation using m7G-specific antibody, purified on RNA Clean & Concentrator - 5 columns (Zymo) and quantified by scintillation counting (Hidex 300 SL).