Neb lunascript rt supermix kit

Reverse transcription for qPCR was either performed using gene-specific reverse priming with the iScript^™ Select cDNA Synthesis Kit (Bio-Rad #1708897) or using NEB LunaScript RT SuperMix kit (NEB #E3010L). In both cases, manufacturer protocols were followed using an input of 2.5 ng of RNA per μL of reaction. For gene-specific priming, the reverse primer was used at 5 μM. The IDT Primetime gene expression master mix (IDT #1055771) was used for quantitative PCR on a Bio-Rad CFX384 instrument with Taqman probes (1.5 μM for primers; 600 nM probe). For samples with spike-ins, abundances were calculated relative to the spike-in abundance using the ΔΔCq method.

RNA extracts from wastewater samples were used to produce amplicons and to prepare libraries according to the COVID-19 ARTIC v3 protocol²⁴ with minor modifications. Briefly, extracted RNA was reverse transcribed using the NEB LunaScript RT SuperMix kit (New England Biolabs) and the resulting complementary DNA was amplified with the ARTIC v3 panel from IDT. ARTIC primers used were: ARTIC V4.1 NCOV-2019 Panel, 500rxn 10011442, IDT ARTIC v3 panel 500rxns 10006788 (IDT). The amplicons were end-repaired and polyadenylated before ligation of adapters using NEB Ultra II (New England Biolabs). Fragments containing adapters on both ends were selectively enriched and barcoded with unique dual indexing with PCR. Libraries were sequenced using the Illumina NovaSeq 6000 and MiSeq platforms, resulting in paired-end reads of 250 bp length each (see Supplementary Information for quality metrics of the sequencing data).