Synthesis of Engineered mRNAs for Multiplex Studies

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.