mRNA Purification from dsRNA Contaminants

To digest dsRNA contaminants present in the IVT mRNA sample, an aliquot of 2.5 μL of RNase III (Epicentre) diluted to 0.01 IU/μL in reaction buffer (33 mM of Tris, pH 8.0, 200 mM of potassium acetate, and 1 mM of magnesium acetate) was combined with 100 μg of mRNA in a final volume of 125 μL of reaction buffer and incubated at 37°C for 30 min.³⁹ Following a phenol-chloroform (pH 4.5; Thermo Fisher Scientific) and two chloroform extractions, the mRNA was precipitated from the aqueous phase by adding one tenth volume of 3 M sodium acetate, pH 5.5, and an equal volume of isopropanol. The centrifuged pellet was reconstituted in water and stored at −20°C. Verification of removal of dsRNA was completed using immunoblot assay, as previously described^{35 (link)} and shown in Supplementary Fig. S1.