Dneasy blood and tissue kit protocol

Vector genome copy determination of the transduced cells was performed by first isolating DNA using the DNeasy Blood and Tissue kit protocol (QIAGEN, Venlo, the Netherlands). DNA was eluted in 50 μL of water for injection, and the concentration was measured by NanoDrop 2000 (Thermo Fisher Scientific, Loughborough, UK). Primers specific for the CAG promoter were used to amplify a sequence specific for the transgenes by SYBR Green Fast qPCR (Thermo Fisher Scientific). The vector genome copies per microgam of genomic DNA input of the samples were calculated by interpolation of a standard line of the expression cassette. To define the background levels of the qPCR, a blank sample was subjected to qPCR using the same expression cassette-targeting primers.

To quantify the amount of DNA, exosomes were resuspended in 200 μL 1x PBS. DNA was extracted following DNeasy Blood and Tissue kit protocol (Qiagen, Hilden, Germany). Quantification was performed on the Synergy H4 Hybrid Microplate Reader (Biotek, Winooski, VT) using the Take 3 microvolume plate and Gen5 software (Biotek). We corrected for the blank and DNA concentration was calculated based on the absorbance ratio of 280/260.

The Qiagen DNeasy blood and tissue kit protocol was used, and the DNA was eluted in 50 μL elution buffer (AE). The DNA samples were sent to Novogene for library preparation, sequencing (Illumina NovaSeq 6000), and raw read filtering (BioProject accession no. PRJNA853708).

The 12 M. viscosa strains analysed here include representatives isolated from different fish species that span the geographical area of occurring outbreaks of winter-ulcer disease across the North Atlantic region (Additional file 1: Table S1). The isolates include both typical and variant M. viscosa, which were categorized as per standard biochemical and phenotypic methods as well as sequence analysis [2 (link), 5 (link), 8 (link), 21 (link)]. The complete genome of the virulent M. viscosa MV 0609139 [22 (link), 23 ] was used as reference. Strains were cultured in Luria-Bertani broth containing 3.5% NaCl at 12 °C. DNA was extracted using the Qiagen DNeasy blood and tissue kit protocol for Gram-negative bacteria.

PAO1 WT genomic DNA was isolated as specified in the Qiagen DNeasy blood and tissue kit protocol. Three hundred nanograms of DNA was PCR amplified using the cloning primers PA0805.1 F and PA0805.1 R described in Table S5. The PCR product was gel extracted with the GeneJet gel extraction kit (Thermo Fisher) and TOPO cloned (Invitrogen). The TOPO reaction was transformed into Escherichia coli TOP10 and selected with 50 μg/ml of kanamycin (TOPO). Plasmid was subsequently isolated according to instructions with the Thermo Fisher kit and digested with the restriction endonucleases EcoRI and KpnI. After the fragment was gel extracted, it was ligated into the similarly digested vector pHERD20T with T4 DNA ligase (Thermo Scientific), transformed into TOP10 E. coli, and selected with 100 μg/ml of ampicillin. Plasmid sequences were confirmed by Sanger sequencing at the UBC Sequencing and Bioinformatics Consortium.

Sampling was performed to represent diverse Nigerian agro-climatic zones. From each zone, two villages were selected and from each village 4–10 scavenging chickens were sampled by drawing blood (50–250 μl) from the wing vein with the logistical support and agreement of the Department of Animal Sciences, Obafemi Awolowo University (Ife Ife, Nigeria). All animal works were approved by the Institutional Animal Care and Use Committee of the International Livestock Research Institute (IREC2017-26) and were handled strictly in compliance with the guidelines of this committee. A geographic coordinate (latitude and longitude) was collected for each sampled village for the extraction of environmental data from public databases.
All the collected blood samples were processed for DNA using the Qiagen DNeasy blood and tissue kit protocol (https://www.qiagen.com/ca/resources/download.aspx?id=63e22fd7-6eed-4bcb-8097-7ec77bcd4de6&lang=en). The genomic DNA (gDNA) from each sample was then normalized to a final volume of 100 µl and final concentration of 50 ng/µl and was sent to Edinburgh Genomics (http://genomics.ed.ac.uk/) in the UK for whole-genome sequencing.

Raw BALs supernatants were spun at 10,000g and frozen at −80 °C. Pellets were resuspended in 180 μl of freshly prepared lysozyme solution (20 mg/ml lysozyme, 20 mM Tris-Cl, pH 8.0, 2 mM Na-EDTA, 1.2 % Triton X-100, filtered through a 0.22-μm filter) and incubated at 37 °C for 10 min. Extraction followed the Qiagen DNEasy Blood and Tissue kit protocol. Reagent controls, without BAL, were also processed through the Qiagen procedure. Extracted DNA was used in a PCR reaction with Roche/454 GS-FLX sequencing adapter fusion primers targeting the V1–V3 region of the bacterial 16S ribosomal RNA (rRNA). The reaction mix consisted of 0.75 U Accuprime Taq Hifi, 1× Accuprime PCR buffer II, 1 μl DNA sample, and 200 nmol of barcoded primer. Cycling conditions were 95 °C for 2 min, then 30 cycles of 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 5 min. Reactions were cleaned up using 36 μl of Ampure Beads (Agencourt) and eluted into 25 μl low TE, pH 8.0. Eluted PCR products were quantitated in triplicate using DNA Picogreen (Invitrogen). Two final sample pools were created by combining 103 and 104 total PCRs; each positive sample had 20 ng added, while each negative control sample (buffer) had 20 μl added. The combined pools were then purified on a MinElute PCR column (Qiagen) and eluted into TE buffer. A total of 170 BAL samples were multiplexed in this manner in two separate gasket wells.