Nanodrop instrument

The majority of genomic DNA (gDNA) samples (n = 204) were extracted in duplicate with the automated chemagic MSM I system according to the manufacturer’s specifications (PerkinElmer Chemagen Technologie GmbH, Baesweiler, Germany). The remaining gDNA samples (n = 23) were extracted at external labs and sent to us. Genomic DNA integrity and concentration were evaluated on a Nanodrop instrument (Life Technologies, Carlsbad, CA, USA). Aliquots of gDNA were diluted to 10 ng/μL with ddH₂O for use in PCRs.

Fresh young leaves of Z. aethiopica and Z. odorata were collected from the Garden Greenhouse (Geospatial coordinates: 102.83945, 24.88627), College of Landscape and Horticulture, Yunnan Agricultural University (Kunming, Yunnan Province, China). High-quality genomic DNA was extracted using the CTAB method [38 (link)]. DNA sample quality was evaluated with agarose gel electrophoresis, and the concentration was measured using a Nanodrop instrument (Thermo Fisher Scientific, Waltham, MA, USA, 2000c UV-Vis). The qualified samples were sent to Wuhan GrandOmics Technology Co., Ltd. (http://www.grandomics.com; accessed on 12 December 2022) for Illumina sequencing and Oxford Nanopore sequencing.

Enteroid monolayers were respectively infected with PEDV (MOI = 5), TGEV (MOI = 1), and PDCoV (MOI = 2), or mock-infected with DMEM-F12 for 24 h and were harvested for RNA-Seq as previously described (18 (link)). Briefly, total RNA was extracted from infected and uninfected enteroids using the TRIzol reagent according to standard protocols. RNA purity and integrity were assessed by an Agilent 2100 Bioanalyzer (Agilent Technologies), a NanoDrop instrument (Thermo Fisher Scientific), and 1% agarose gel electrophoresis, and a total amount of 3 μg RNA per sample was used for library preparation using the NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, USA) following manufacturer’s recommendations. The library preparations were sequenced on the Illumina HiSeq 4000 platform (Illumina). All sequences were processed and aligned to the Sus scrofa reference genome. The Deseq2 R package was used to determine differentially expressed genes at a significance cutoff of P value < 0.05. The differentially expressed genes (DEGs) were selected based on the screening criteria that | log₂ (fold change) | > 1 and false discovery rate (FDR) < 0.05.

Rice samples were planted at a greenhouse in CAAS. Five plants growing normally at the same stage (3–5 leaves) were selected randomly from G2-6 and ZH11 for the extraction of total genomic DNA. Total genomic DNA of rice leaves was extracted using a DNA extraction kit. To produce spike-in positive control samples for sequencing, the plasmid DNA was added to the genomic DNA of ZH11 at one copy per genome equivalent. DNA quantity was evaluated by 1% agarose gel electrophoresis and a NanoDrop instrument (Thermo Scientific, United States).

Aliquots of clinical samples were processed as follows: 600-μl aliquots of Aptima samples, 300-μl aliquots of HC2 samples and 4-ml aliquots of ThinPrep samples were extracted using the miRCURY RNA Isolation Kit—Cell and Plant (Exiqon A/S, Vedbaek, Denmark) applying the preparation protocol for nasal or throat swabs. For HC2 and ThinPrep samples the cells were first centrifuged down (5 min 12,000g for HC2 and 15 min 2900g for ThinPrep), after which the supernatant was removed and 600 μl of lysis solution was added. After 5 min incubation at room temperature, 600 μl of 70 % ethanol was added and the extraction was carried out according to the manufacturer’s purification protocol for total RNA. 600 μl of 70 % ethanol was added directly to Aptima sample aliquots without adding lysis solution. Total RNA was eluted in 50 μl of elution buffer and stored at −70 °C until reverse transcription was performed. From each FFPE sample four 20-μm sections were prepared and RNA was isolated using the RecoverAll total RNA isolation kit (Ambion, Austin, TX). Total RNA from cultured cells was isolated using the mirVana RNA isolation kit (Ambion). RNA concentrations were measured in NanoDrop instrument (Thermo Scientific, Wilmington, DE).