Sybr green 1 solution

Total DNA from hypocotyl samples was extracted using a cetyltrimethylammonium bromide (CTAB) method [38 (link)]. About 100 mg of ground fresh plant tissue was suspended in 1 ml CTAB with 2 µl ß-mercaptoethanol and 1 µl 1x proteinase K. The extracted DNA was dissolved in 200 µl TE buffer.
A CFX384 real-time PCR detection system (Bio-Rad Laboratories Inc., Kabelsketal, Germany) was used for the amplification and quantification of V. longisporum DNA using primers OLG70 (5′-CAGCGAAACGCGATATGTAG-3′) and OLG71 (5′-GGCTTGTAGGGGGTTTAGA-3′) [39 (link)]. The amplification mix consisted of 1× (NH₄)₂SO₄ buffer, 2.5 mM of MgCl₂, 100 µM of dNTPs, 0.02 U/µl of BioTaq DNA polymerase (Bioline, Luckenwalde, Germany), 0.1x SYBR Green I solution (Invitrogen, Karlsruhe, Germany), 0.3 µM each of primers OLG70 and OLG71 and 1 µl of template DNA and filled up to a total volume of 10 µl with ddH₂O. PCR conditions were as described in Table 3. PCR for all treatment samples were performed with four biological and three technical replicates and data were analyzed using CFX Manager Software.

Candida cells at logarithmic phase of growth (1 × 10⁶ cells/mL) were treated with NPV or NMV (1, 10, 100 and 1000 µg/mL) for 2 h; the cells were then washed, resuspended in TE buffer (50 mM Tris-HCl, pH 8 containing 50 mM EDTA) and fixed using 95% ethanol. Cells were then washed using TE buffer and treated with 1 mg/mL RNAse A at 37 °C for 1 h and then treated with 5 mg/mL Proteinase K at 37 °C for 1 h. Cells were then washed using TE buffer and resuspended in a SYBR Green I solution (1:85 dilution of a commercial stock in TE buffer, Thermo Fisher Scientific, Waltham, MA, USA) and incubated overnight at 4 °C. Stained cells were then resuspended in TE buffer, and the phases of cell cycle (G1, S and G2/M) were analyzed using an Amnis^® FlowSight^® (Merck KGaA, Darmstadt, Germany) imaging flow cytometer and IDEAS software version 6.2.187.0 (Merck KGaA, Darmstadt, Germany). Representative histograms (normalized frequency vs intensity) are presented.

Evaluation of the cfDNA concentration was also performed measuring the fluorescence of intercalating dye [24 (link)]. Specifically, 5 μL of a sample or the same volume of a standard dilution of genomic DNA (Human HCT116 DKO Nonmethylated DNA) with known concentration (0 ng/mL and 9 serial dilutions from 1 to 256 ng/mL) was added to 195 μL of a SYBR Green I solution (Cat. number S7585, Thermo Fisher Scientific, USA) in PBS buffer (1 : 10,000) and to black 96-well plates (PAA, Cat. number PAA30296X, Austria) and incubated for 10 min. Two to three identical mixtures were prepared from each sample or standard for greater accuracy. The fluorescence of the mixtures obtained was measured by the “VICTOR³ 1420-050” Multilabel Plate Readers (Perkin Elmer, USA) using filters for FITC (485/535 nm) and 1 s acquisition time. The DNA concentration was calculated from the standard curve (R² was 0.97).

Cells were treated with 1 mg/mL RNAse A at 37°C for 1 h followed by 5 mg/mL Proteinase K at 37°C for another 1 h. After washing with TE buffer, cells were resuspended in a SYBR Green I solution (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight. Flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) and the IDEAS software version 6.2.187.0 (Merck KGaA, Darmstadt, Germany) were used to determine the phases of the cell cycle.

Bacteria and VLPs were enumerated using an Accuri C6 flow cytometer (Becton Dickinson), which was fitted with a 488 nm blue laser and green (530/30 nm), orange (585/40 nm) and red (670 nm) filters. Prior to analysis, triplicate samples were thawed and diluted to 1:10 with 0.2 μm filtered TE buffer (10mM Tris, 1 mM EDTA, pH 8). Samples were then stained with SYBR Green-I solution (1:20000 dilution; Molecular Probes, Eugene, OR) and incubated in the dark at 80°C for 10 min [26 (link)]. As an internal size and fluorescence standard, 1 μm diameter fluorescent beads (Molecular Probes, Eugene, OR) were added to each samples at a final concentration of approximately 10⁵ beads ml^-1 [29 ]. For each sample, forward scatter, side scatter and green (SYBR Green-I) fluorescence were acquired for two minutes. FlowJo (Treestar, Inc.) software was used to analyse data collected from each sample, where differences in cell side scatter and SYBR Green fluorescence were used to discriminate between VLP and bacterial groups [26 (link),30 (link),31 (link)].

Cell numbers in extracted soil communities and in the mixed liquid suspended growth experiments were counted by flow cytometry. SC-suspensions were diluted 100 times in MMS and stained in 200 µl aliquots with 2 µl of diluted SYBR Green I solution (1:100 in DMSO; Molecular Probes) in the dark for 30 min at room temperature. In some experiments, cells were additionally stained with 2 µl propidium iodide solution (10 µg ml^–1, Molecular Probes). Aliquots of 20 µl were aspired at 14 µl min^–1 on a Novocyte flow cytometer with absolute volumetric cell counting (ACEA Biosciences, USA). Cells were thresholded above a forward scatter signal (FSC-H) of 20 and further gated for propidium iodide-staining (excited at 535 nm and its fluorescence was collected at 617 ± 30 nm) and for SYBR Green I (excitation 488 nm, 530 ± 30 nm band-pass filter; channel voltage at 441 V) above values of 1000 (Supplementary Fig. 5).
Cell samples from the mixed liquid suspension growth experiments were diluted to approximately 10⁶ ml⁻¹ and subsampled to aliquots of 100 µl. The subsamples were then mixed with 100 µl of 8 g l^–1 sodium azide in phosphate buffered saline and incubated for 1 h at 4 °C to arrest cell respiration and growth. Samples were then stained with SYBR Green I as above and quantified by flow cytometry using the same thresholds and gates as describe above.

The yeast and 14 bacterial pure cultures that were used as standards in building ANN classifiers are listed in Table 1. The strains were initially selected on the basis of their occurrence in aquatic systems, and with the aim of including variation in size and morphology (e.g., rod, coccus), as well as groups of strains with similar taxonomy (e.g., Pseudomonas, Arthrobacter, Sphingomonas). Strains were grown aseptically and individually in liquid media in biological triplicates until reaching stationary phase (conditions provided in Supplementary Table 1). For E. coli, we further included samples from exponential growth (OD₆₀₀ = 0.5) and from stationary phase (OD₆₀₀ = 2) on two different culture media (Supplementary Table 1). Culture samples were diluted in phosphate-buffered saline (PBS) to 10⁵ or 10⁶ cells ml^–1 and stained in 200 µl aliquots with 2 µl of diluted SYBR Green I solution (1:100 in dimethylsulfoxide; Molecular Probes) in the dark for 15–30 min at 20 °C for FCM analysis.
Bead standards consisted of polystyrene size calibration beads with diameters of 0.2, 0.5, 1, 2, 4, 6, 10, and 15 µm (Invitrogen), used in solution at concentrations of 1 × 10⁶ (0.2 and 0.5 µm), 6 × 10⁷ (1 µm), 3 × 10⁷ (2 and 4 µm) and 2 × 10⁷ (6, 10, and 15 µm) beads ml^–1. Beads were stored and prepared for FCM analysis according to the manufacturer’s guidelines.