Neb 5 alpha competent escherichia coli cells

NEB 5-alpha competent Escherichia coli cells (New England BioLabs, Hitchin, UK) were transformed with the vector according to the manufacturer’s instructions. Briefly, 10 ng of plasmid DNA was carefully mixed with cells and incubated on ice for 30 min. Cells were heat shocked at 42 °C for 30 s and chilled on ice for 5 min. After the addition of 950 µL of SOC medium (Sigma Aldrich, St. Louis, MO, USA), the mixture was shaken at 200 rpm for 60 min at 37 °C, and the cells were diluted and plated on selective LB agar (Invitrogen, Waltham, MA, USA) plates containing 100 µg/mL ampicillin (Gibco, Waltham, MA, USA). After incubation overnight at 37 °C, selected colonies were inoculated into 3 mL of fresh LB medium containing 100 µg/mL ampicillin and incubated overnight at 37 °C with shaking at 200 rpm. The vector construct with the RdRp sequence was verified by Sanger sequencing. Plasmids for subsequent transfection of lung cells were purified using a PureLink HiPure Plasmid Miniprep Kit (Invitrogen, Waltham, MA, USA).