Tianamp virus rna extraction kit

RNA was extracted from the P3 generation, P6 generation, P9 generation, P12 generation, and P15 generation of recombinant RABV, rSRV9-H, and rSRV9-F using a TIANamp virus RNA extraction kit (Tiangen, Beijing, China). After transcribing the RNA into cDNA using TransScript One-Sep RT–PCR SuperMix (TransGen, Beijing, China), identification was performed via PCR using the primers listed in Table 1. The sizes of the bands were analyzed, and gel blocks containing DNA of the correct size were sent to Comate Bioscience (Jilin, China) for sequencing.

A detailed procedure for the amplification and sequencing can be found in Supplementary Methods. Briefly, the total vRNA of each resistant supernatant was extracted using a TIANamp Virus RNA extraction Kit (Tiangen Biotech, Beijing) following the manufacturer’s instructions. The genomes were amplified using a SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase (Invitrogen, Carlsbad, CA). The fragments were checked by agarose gel electrophoresis and retrieved by gel extraction using a TIANgel Midi Purification Kit (Tiangen Biotech, Beijing), following the manufacturer’s instructions. An aliquot of each fragment was sequenced (Sangon Biotech, Shanghai), and the sequences were analyzed using the software Lasergene SeqMan Pro v.7.1 (DNASTAR, Madison, WI).