Protocol to isolate temperature-sensitive SARS-CoV-2 mutants and identify associated mutations

An inability to proliferate at high temperatures typically gives viruses an attenuated phenotype. Here, we present a protocol to obtain and isolate temperature-sensitive (TS) SARS-CoV-2 strains via 5-fluorouracile-induced mutagenesis. We describe steps for the induction of mutations in the wild-type virus and selection of TS clones. We then show how to identify the mutations associated with the TS phenotype, following forward and reverse genetics strategies.