The sequences of the primer and template used for these extension reactions, which are at the 3’ end of the SARS-CoV-2 genome, are shown at the top of the figure. Polymerase extension reactions were performed by incubating (a) 2’-F,Me-UTP, (b) 3’-F-dTTP, (c) UTP + TFV-DP, and (d) 3’-N3-dTTP with pre-assembled SARS-CoV-2 polymerase (nsp12, nsp7 and nsp8), the indicated RNA template and primer, and the appropriate reaction buffer, followed by detection of reaction products by MALDI-TOF MS. The detailed procedure is shown in the Methods section. The accuracy for m/z determination is ± 10 Da.