Betamercaptoethanol

On procedure days, 100 μL of blood from K2-EDTA collection tubes was collected prior to centrifugation and was added to 600 μL of AVL viral lysis buffer with 6 μL carrier RNA (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 mL RNAlater (Qiagen) for at least 4 days for stabilization. RNAlater was completely removed, and tissues were homogenized in 600 μL RLT buffer and 1% betamercaptoethanol (Qiagen) in a 2 mL cryovial using a TissueLyser (Qiagen) and 0.2 mm ceramic beads. The tissues sampled included axillary and inguinal lymph nodes, liver, spleen, kidney, adrenal gland, lung, pancreas, urinary bladder, ovary or testis, and eye. All blood samples were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood using the QIAamp viral RNA kit (Qiagen), and from tissues using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit.

Lung tissue samples were weighed and then added to reinforced PreCellys tubes containing ceramic beads (Stretton Scientific, Alfreton, UK). Samples for RNA extraction were homogenised with RLT buffer supplemented with 1% (v/v) beta-mercaptoethanol (QIAGEN, UK) using an automated PreCellys21 homogeniser (Stretton Scientific, UK). Samples for the focus-forming unit assay were homogenised with PBS. Non-tissue samples for RNA extraction were inactivated in AVL buffer (QIAGEN) and ethanol. Downstream RNA extraction on all samples was performed using the BioSprint One-For-All Vet Kit (Indical, UK) and the Kingfisher Flex Platform (Thermo-Fisher, UK) as per the manufacturers’ instructions.

A FACS Aria III cell sorter (Becton Dickinson, San Jose, USA) equipped with a 488 nm blue solid laser was used for analysis. Forward-scatter characteristics (FSC) resulted from the small-angle scatter, while side-scatter characteristics (SSC) were recorded as orthogonal scatter of the 488 nm laser. Fluorescence (eYFP/Venus) was detected by a 502-nm long-pass and a 530/30 nm band-pass filter set. The FACS software DIVA 7.0 was used for the recording. Thresholding on the FSC/SSC was applied for all measurements and removed for the sorting procedure. Initially, to enable gate settings uninfected kidney cell suspension were analysed. Cell sorting was achieved by pre-gating the cells twice. FSC and SSC characteristics were used to set the first gate to remove non-cell particles. The next gate was set around the FSC height and fluorescence area signal to select stained population of cells. From these pre-gating settings, the cells were sorted with the four-way purity mask and a threshold rate of 8000–25 000 events/s with 70 μm nozzle. Cells were sorted directly into 1.5 mL Eppendorf tubes containing cold lysis buffer with beta-mercaptoethanol (Qiagen, Germany) and the sorted cells were stored immediately at −80 °C. The parasite viability was examined visually under the fluorescence microscope before and after cell sorting.