RNA Isolation and Directional Library Prep

RNA isolation and library preparation is fully described in Butler, et al. (Butler et al., 2021 (link)). Briefly, library preparation on all the nasopharyngeal swab samples’ total nucleic acid (TNA) were treated with DNAse 1 (Zymo Research, Catalog # E1010). Post-DNAse digested samples were then put into the NEBNext rRNA depletion v2 (Human/Mouse/Rat), Ultra II Directional RNA (10 ng), and Unique Dual Index Primer Pairs were used following the vendor protocols from New England Biolabs. Kits were supplied from a single manufacturer lot. Completed libraries were quantified by Qubit or equivalent and run on a Bioanalyzer or equivalent for size determination. Libraries were pooled and sent to the WCM Genomics Core or HudsonAlpha for final quantification by Qubit fluorometer (ThermoFisher Scientific), TapeStation 2,200 (Agilent), and qRT-PCR using the Kapa Biosystems Illumina library quantification kit.