Cleancap reagent ag

mRNA was produced using standard in vitro transcription, according to previously established methods [19 (link)] and the manufacturer’s recommendations. Briefly, plasmid DNA encoding firefly luciferase with poly-A tail was digested by restriction enzyme Sap I, followed by the production of mRNA using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) with CleanCap^® Reagent AG (TriLink BioTechnologies, NorthPark, CA, USA), according to the manufacturer’s instructions. Thereafter, mRNA was purified.

Murine Fam64a-FLAG or EGFP sequence downstream of T7 promoter was cloned into pGEMHE, and was PCR amplified using a primer set (3′-GTAAAACGACGGCCAGT-5' and 3′-CAGGAAACAGCTATGAC-5′). This PCR product was purified using FastGene Gel/PCR Extraction Kit (Nippon Genetics, Japan), and was used as the template for modified mRNA synthesis. In vitro transcription was performed using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, USA) with a customized ribonucleoside blend of GTP (1.5 mM), ATP (7.5 mM), CTP (7.5mM), N1-Methylpseudo-UTP (7.5 mM, N-1081, Tri-Link Biotechnologies), and CleanCap® Reagent AG (6 mM, N-7113, Tri-Link Biotechnologies, USA) (Zangi et al., 2017 (link); Kaur and Zangi, 2020 (link)). Following the purification of transcribed mRNA using Fast Gene RNA Premium Kit (Nippon Genetics), Poly (A) tailing reaction was performed using E. coli Poly (A) polymerase (New England Biolabs), and mRNA was re-purified with the same kit. The size and the integrity of synthesized modified mRNA was checked by agarose gel electrophoresis, and quantity was determined using a NanoDrop One spectrophotometer (Thermo Scientific).

mRNAs were synthesized as previously described (94 (link), 95 (link)). Briefly, linearized plasmids encoding firefly luciferase, enhanced GFP, and mCherry were transcribed using the MEGAscript T7 Transcription Kit (Ambion) with cotranscriptional capping with CleanCap reagent AG (TriLink Biotechnologies). One-methylpseudouridine (m1Ψ)-5′-triphosphate (TriLink) instead of uridine 5′-triphosphate was used to generate modified nucleoside-containing mRNA. Double-stranded RNA (dsRNA) contaminants were removed by cellulose purification as described (96 (link)). Briefly, 700 μg of crude mRNA was loaded into a spin column containing 700 μl of 16% cellulose (w/v). Columns were incubated at room temperature with constant shaking for 25 min. dsRNA-free mRNA was collected in the flow-through and then purified a second time. All mRNAs were analyzed by electrophoresis using agarose gels and confirmed to be free of dsRNA contaminants by dot blot (J2 monoclonal antibody, Abcam) and lack of TNF-α production by primary human dendritic cells (Human Immunology Core, University of Pennsylvania). In vitro transcribed mRNAs were used for multiplexing and luciferase immunohistochemistry studies.