Qiaamp mini column

Total RNAs from urine supernatants were extracted using TRIzol® reagent (QIAamp Mini column, Qiagen, USA) according to manufacturer instructions. The concentration of the extracted RNA was measured using NanoDrop spectrophotometer.

For each sample or blank control, 1 mL of the final sample suspension, mixed with the InhibitEX buffer, was transferred into a 2 mL microcentrifuge tube. For membrane filtered samples and controls, microcentrifuge tubes with the filter and 1 mL of InhibitEX buffer were used. After 1 min of vortexing, the solution was centrifuged at maximum speed for 2–3 min, and the suspension was incubated at 95°C for 5 min, followed by full speed microcentrifugation for 15 s to pellet the sample particles. Subsequently, 600 μL of the supernatant was transferred into a 2 mL microcentrifuge tube containing 25 μL proteinase K, and then 600 μL of buffer AL was added. After vortexing for 15 s, the sample was incubated at 70°C for 10 min, and 600 μL of 100% ethanol was added to the lysate. A 600 μL volume of lysate was applied to a single QIAamp Mini column (Qiagen, Cat. # 51604, Hilden, Germany), and the column was centrifuged at full speed for 1 min. This procedure was repeated two additional times with the same column in order to use up all of the lysate. Finally, DNA was eluted from the column with 150 μL of the supplied elution buffer. The extracted DNA samples were aliquoted and stored at −80°C until analyzed by qPCR.

For F2 pooled genotyping, genomic DNA was extracted from ~20mg at the anterior end of each adult fluke using the DNeasy Blood and Tissue kit (Qiagen, UK) with elution in 100μl of buffer AE. This was followed by precipitation using 3M NaOAc and isopropanol at 4°C. Each individual fluke DNA was checked for quality on a 2% agarose gel and quantified by Quant-IT PicoGreen (Life technologies/ThermoFisher Scientific). Equimolar concentrations of genomic DNA from each parasite was mixed and purified with GenomicTip (Qiagen, UK) to create an F2 pool of high molecular weight DNA per sheep, for sequencing. Egg DNA from field isolates was extracted using the DNeasy Blood and Tissue kit (Qiagen, UK) with the following modifications: (i) a micropestle (Argos Technologies, USA) and Pellet Pestle Motor (Kontes) were used to homogenise the eggs; (ii) RNase A was used; (iii) elution was in 100μl of buffer AE. 5μl of egg DNA was subjected to whole genome amplification using a REPL-g Mini Kit (Qiagen, UK), followed by purification using QiaAmp Mini Column (Qiagen, UK) with the following modifications (i) the DNA was added to the spin column and then the wash steps were performed (ii) elution was with 65μl of buffer AE.