Viral Capture of SARS-CoV-2 from Rhesus macaque
cDNAs were prepared according to Briese et al, with minor modifications.  Briefly, 3 to 12 l of extracted RNA was depleted of rRNA using Ribo-Zero Gold H/M/R (Illumina, San Diego, CA) and then reverse-transcribed using random hexamers and SuperScript IV (ThermoFisher Scientific, Waltham, MA).  Following RNaseH treatment, second strand synthesis was performed using Klenow fragment (New England Biolabs, Ipswich, MA) and resulting double-stranded cDNAs were treated with a combined mixture of RiboShredder RNase Blend (Lucigen, Middleton, WI) and RNase, DNase-free, high conc (Roche Diagnostics, Indianapolis, IN)  and then purified using Ampure XP bead purification (Beckman Coulter, Pasadena, CA).  Kapas HyperPlus library preparation kit (Roche Sequencing Solutions, Pleasanton, CA) was used to prepare sequencing libraries from the double-stranded cDNAs.  To facilitate multiplexing, adapter ligation was performed with KAPA Unique Dual-Indexed Adapters and samples were enriched for adapter-ligated product using KAPA HiFi HotStart Ready mix and 7 PCR amplification cycles, according to the manufacturer?s manual.  Pools consisting of eight sample libraries were used for hybrid-capture virus enrichment using myBaits Expert Virus SARS-CoV-2 panel and following the manufacturers manual, version 4.01, with 14 cycles of post-capture PCR amplification (Arbor Biosciences, Ann Arbor, MI).