Rneasy powermicrobiome rna isolation kit

The NetoVIR protocol was used to process the enrichment of the viral particles for all twelve faecal samples [20 (link)], albeit with some in-house modifications. Briefly, 10% faecal suspensions were prepared in phosphate buffered saline (PBS), followed by homogenization and centrifugation. The supernatant was filtered through a 0.45 µm filter to remove eukaryotic and bacterial cell-sized particles but to retain larger RNA viruses. The resulting filtrate was subjected to nuclease treatment using Benzonase Nuclease (Sigma-Aldrich, St Louis, MO, USA) and Micrococcal Nuclease (Thermo Scientific, Waltham, MA, USA) in a buffer consisting of 1 M Tris, 100 mM CaCl₂, and 30 mM MgCl₂, pH 8, for 2 h, at 37 °C, to digest unprotected nucleic acids. The nuclease enzymes were inactivated with EDTA. Extraction of viral RNA was performed on viral particle enriched samples using a RNeasy PowerMicrobiome RNA isolation kit (Qiagen, Hilden, Germany). However, the bead-based homogenization step was bypassed since the enriched sample was already in liquid form. The eluted, purified, and quantified RNA was subjected to host ribosomal RNA (rRNA) removal using a NEBNext ribosomal RNA (rRNA) depletion (Human/Rat/Mouse) kit (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s protocol.

In the site-scale study, RNA was extracted from three parallel sediment cores from both the riparian zone and riverbed sediments using an RNeasy Power Microbiome RNA Isolation Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. RNA quality and concentration were estimated using a NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
Reverse transcription was performed with a PrimeScript^TM RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China). All RT-qPCR analyses were performed on a sequence detection system (ABI 7500; Applied Biosystems, Foster City, CA, USA) with SYBR-Green fluorescent dye (TaKaRa). The copy numbers of N₂O-related genes were quantified by using the specific primers for the archaeal amoA gene with Arch-amoAF and Arch-amoAR, the bacterial amoA gene with amoA-1F and amoA-2R, the denitrifier and ammonia oxidizer nirK gene with nirK-876F and nirK-1040R, the nirS gene with nirS-F and nirS-R, the denitrifier and ammonia oxidizer norB gene with norB-F and norB-R, and the nosZ gene with nosZ-F and nosZ-R. All tests were conducted in triplicate with amplification efficiencies between 90% and 110% and correlation coefficients (R²) above 0.98. More details on the primers and thermal profiles are listed in Supplementary Table S4.