Viral RNA Extraction from Sewage Samples

Viral RNA was extracted from each sewage sample after centrifugation, to produce a solid fraction, and after PEG precipitation of the supernatant, to produce a PEG-precipitated fraction, following the procedures of previous studies with minor modifications (Jones and Johns, 2009 (link); Kitamura et al., 2021 (link)). Specifically, 160 ml of each sample was divided equally into four aliquots (40 ml each) held in 50 ml tubes and centrifuged at 10,000 × g for 30 min. RNA was extracted from the resulting pellet (solid fraction sample) using the NucleoBond RNA Soil kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. Meanwhile, the entire supernatant was precipitated using PEG 8000 (final concentration 10%; Promega, Madison, WI, United States) and NaCl (final concentration 1 M; Wako, Tokyo, Japan) by incubating at 4°C overnight with gentle rotation. After centrifugation at 10,000 × g for 60 min, the precipitate was resuspended in 500 μl of phosphate buffer solution (pH 7.0, 0.067 mol/L; Nacalai Tesque, Kyoto, Japan). RNA was extracted from 140 μl of the PEG-precipitated suspension using a QIAamp Viral RNA Kit (Qiagen, Hilden, Germany). RNA was also extracted from 140 μl of raw unconcentrated sewage samples using a QIAamp Viral RNA Kit (Qiagen).