Superscript 4 rt cdna synthesis kit

We sequenced the entire N gene in 207 samples by Sanger sequencing. cDNA was prepared using a SuperScript IV RT cDNA synthesis kit (Thermo Fisher Scientific Inc. (Invitrogen), Waltham, MA, USA). Gene N was amplified using 3 sets of primers generating amplicon lengths of about 500 nts with 50 nt overlaps. The sequencing primers of set 1 for the N gene are the following: CovNg-1F 5′-CATGACGTTCGTGTTGTTTTAGAT-3′, CovNg-1R 5′-GCCAATGTGATCTTTTGGTGTATT-3′. Set 2 for the N gene: CovNg-2F 5′-GAATACACCAAAAGATCACATTGGCA-3′, CovNg-2R 5′-TCCTTGTCTGATTAGTTCCTGGTCC-3′. Set 3 for the N gene: CovNg-3F 5′-AAACGTACTGCCACTAAAGCATACA-3′, CovNg-3R 5′-TTATATAGCCCATCTGCCTTGTGT-3′. Gene E was amplified, applying 1 set of primers as follows: CovEg-F 5′-GACTACTAGCGTGCCTTTGTAAGC-3′, CovEg-R 5′-CTGCCATGGCTAAAATTAAAGTTC-3′. Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific Inc. (Invitrogen), Waltham, MA, USA) was used. The sequence of the Wuhan-Hu-1 SARS-CoV-2 isolate was used as the reference genome (NCBI GenBank NC_045512.2). Sequencing was performed using a BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc. (Invitrogen), Waltham, MA, USA). All procedures were performed according to the manufacturer’s instructions.