RNA extraction and quantitative RT-PCR
RNA was extracted from cell culture supernatants and cells lysates using
the ExtractRNA Reagent (Eurogen, Moscow, Russia) following the
manufacturer’s instructions. Reverse transcription and PCR amplification
were carried out with a one-step SARS-CoV-2 FRT kit (N.F. Gamaleya
NRCEM, Moscow, Russia) according to the manufacturer’s instructions. The
primers and probes were designed to target the gene coding NSP1 (Leader
Protein) of SARS-CoV-2 (Fig.1d). The conditions of the one-step RT-qPCR
reaction were as follows: 50°C for 15 min, 95°C for 5 min, followed by
45 cycles of 95°C for 10 s and 55°C for 1 min. The number of copies of
viral RNA was calculated using a standard curve generated by
amplification of plasmid DNA template.