Genome sequencing of SARS-CoV-2 virus stocks
PCR primers spanning the whole genome of SARS-CoV-2 were designed yielding in about 2kb amplicons (Table S9). 2.5 L of RNA were used in three separate RT-PCR reactions as described above with oligonucleotide primers at 400 nM concentration with the following cycling conditions: 55 C for 15 min, 95 C for 3 min; 35 cycles consisting of 95 C for 15 sec and 57 C for 3 min; final extension at 72 C for 10 min. PCR products were combined and purified by incubation with 1.8X Ampure XP beads (Beckman Coulter) followed by two washes with 75% ethanol and elution in 30 L water. Amplicons were fragmented to 150250 bp length and Ion Torrent barcode and sequencing adapters were ligated to the fragments using the NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torren kit (New England Biolabs) according to the manufacturers recommendations. Libraries were sequenced on an Ion Torrent S5XL instrument using a 540 Chip Kit and the 200 bp sequencing kit (ThermoFisher).