Rnase a solution

Genomic DNA was prepared from 25 ml cultures of bacterial isolates grown overnight at 37 °C in LB media. Cells were harvested by centrifugation and resuspended in 2.0 ml of 25% w/v sucrose in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0). Nuclei Lysis Solution (Promega, #A7941, 6.0 ml) was added and samples were lysed by incubation at 80 °C for five minutes. Samples were mixed with proteinase K (250 μg/ml final concentration), RNase A Solution (Promega, #A797A, 15 μg/ml final concentration), EDTA pH 8.0 (25 mM final concentration) and aqueous SDS solution (0.3% final concentration). Mixtures were incubated on ice for two hours and then at 50 °C overnight. Following enzymatic treatment, TE buffer was added to each sample (12 ml final volume). DNA was then isolated by phenol-chloroform extraction. Nucleic acids were precipitated in absolute ethanol, washed in ethanol (70% v/v), and resuspended in approximately 350 μl Tris (10 mM; pH 8.0). EDTA was omitted from the resuspension solution, to avoid interference with PacBio sequencing chemistry.

A single HT115 (DE3) colony was cultured overnight in LB at 37°C with shaking at 220 rpm. The culture was diluted 100-fold in 800 mL 2 × YT supplemented with 75 μg/mL ampicillin and 12.5 μg/mL tetracycline and cultured at 37°C to an OD600 of 0.5. Production of T7 polymerase was induced with 0.4 mM IPTG, and the bacteria were incubated with shaking for an additional 4 hr at 37°C. Total nucleic acids were extracted40 (link). The bacterial pellets were resuspended in 1 M ammonium acetate/10 mM EDTA, and an additional volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added. The samples were incubated at 65°C for 30 min and centrifuged at 12,000 g for 15 min. The upper phase was mixed with isopropanol, incubated at −20°C overnight and centrifuged at 12,000 g for 30 min. The nucleic acid pellet was resuspended in TE. For dsRNA quantification, the nucleic acids were treated with RQ1 RNase-free DNase (Promega, USA) and RNase A solution (Promega, USA). The concentration was determined using a NanoDrop 1000 (Thermo, USA). The dsRNAs were loaded onto a 2% agarose gel, stained with ethidium bromide, and photographed to determine integrity.

PURE reactions (5 μL) labeled with FluoroTect GreenLys (Promega) were incubated with 0.8 μg or 0.2 μL of RNAse A solution (Promega) and incubated for 30 min at 37 °C and subsequently analyzed by SDS-PAGE using 10-well 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad). Gels were scanned (AlexaFluor 488 settings, excitation: Spectra blue 470 nm, emission: F-535 Y2 filter) with a Fusion FX7 Imaging System (Vilber) and analyzed with ImageJ. Protein sizes were calculated based on a BenchMark^TM Fluorescent Protein Standard (Invitrogen).

The l4440 plasmid containing egfp and the target gene fragments was transformed into Escherichia coli HT115 (DE3) competent cells. Modified plasmids were constructed as described by Li³² . After transformation, single colonies of HT115 (DE3) were cultured overnight in LB at 37 °C with shaking at 220 rpm. The culture was diluted 100-fold in 800 ml 2 × YT supplemented with 75 mg/ml ampicillin plus 12.5 mg/ml tetracycline, and cultured at 37 °C until reaching an OD600 value of 0.5. dsRNA synthesis by T7 polymerase was induced by adding 0.4 mM IPTG, and the bacteria were incubated with shaking for an additional 4 h at 37 °C.
Total nucleic acids were extracted as described by Timmons^{33 (link)}. Samples were treated with RQ1 RNase-free DNase (Promega, USA) and RNase A solution (Promega, USA) before measuring concentrations using a NanoDrop 1000 (Thermo, USA). dsRNA solutions were also loaded onto a 2% agarose gel, stained with ethidium bromide, and photographed.

Nucleic acid concentration in nucleic acid eluates was determined via Qubit dsDNA BR and Qubit RNA BR (Invitrogen) assays. If the amount of DNA in the second nucleic acid eluate was insufficient for downstream applications, a fraction of the first eluate was transferred to the second eluate. To prepare DNA for whole-genome sequencing, 1.0 µL of 4 mg/mL RNase A solution (Promega) was added to the DNA followed by a 30-minute incubation at 37 °C. DNA samples were stored at −80 °C.
WGS was performed by Novogene. Briefly, the genomic DNA was sheared into 350 bp fragments, libraries were constructed using the NEBNext DNA Library Prep Kit, followed by end repair, dA-tailing, and ligation with NEBNext adapter. Fragments (300-500 bp) were PCR enriched by P5 and indexed P7 oligos. DNA libraries were checked for quantity and quality using Qubit 2.0 fluorometer and the Agilent 2100 bioanalyzer, respectively. The Illumina Novaseq 6000 (Illumina Inc., San Diego, CA, USA) was utilized for WGS in Novogene Bioinformatics Technology Co., Ltd (Beijing, China) to generate 150 bp paired-end reads.

Retained frozen lysate (50 ml) from strongly positive samples on MC-qPCR (n = 113, Cq value < 30) was thawed at room temperature, centrifuged at 3500 rpm for 15 min, and 250 µl transferred into a 1.5-ml Eppendeorf tube. DNA was extracted using the High Pure PCR Template Preparation kit (Roche, Mannheim, Germany). Protocol followed manufacturer’s instructions except the first step was skipped, as the lysate already contained proteinase K, and 75 µl was used for elution in the final step to increase DNA amount. All samples were extracted in duplicate and treated with 1 µl of RNAse A Solution (4 mg/ml, Promega, Madison, WI, USA). Frozen aliquots of template DNA (50 µl) were sent within 24 h to the Department of Microbiology, University of Tennessee, Knoxville, TN, USA, for further genetic characterization. A Mn-PCR–RFLP method employing ten genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico [44 (link)]) was used as per Su et al. [35 (link)].