Preparation of chimeric recombinant SARS-CoV-2

To investigate the virological properties of a SARS-CoV-2 variant, Omicron BA.2, we generated chimeric recombinant viruses that express GFP and encodes the S gene of B.1.1 (ancestral D614G-bearing virus), Delta, BA.1 and BA.2. To verify the genome sequence of the working viruses, we performed viral RNA-sequencing of the viral stock. Overall design: We prepared the seed recombinant viruses by CPER technique. We then amplified the recombinant viruses using VeroE6/TMPRSS2 cells. At 3 days postinfection, the culture medium was harvested and centrifuged, and the supernatant was collected as the working viruses. The genome sequences of the working viruses were verified using a viral RNA-sequencing analysis. Viral RNA was extracted using QIAamp viral RNA mini kit (Qiagen, Cat# 52906). The sequencing library for total RNA-sequencing was prepared using NEB Next Ultra RNA Library Prep Kit for Illumina (New England Biolabs, MA, Cat# E7530). Paired-end, 76-bp sequencing was performed using MiSeq (Illumina, San Diego, CA) with MiSeq Reagent Kit v3 (Illumina, Cat# MS-102-3001). Contributor: The Genotype to Phenotype Japan (G2P-Japan) Consortium