Ultrapure agarose

Agarose diffusion assays were performed as previously described [77 (link)]. In brief, 5 mm paper disks (Whatmann, Buckinghamshire, UK) were loaded with 5 µL antifungal for the inhibition assay and/or 5 µL modulator for the combination assay and placed on SD pH 6.8 or RPMI1640 growth media solidified with 0.6% UltraPure^TM agarose (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) containing S. cerevisiae or clinical isolates of C. auris inoculated at OD_600nm = 0.08. Control disks contained the same amount of DMSO. Zones of inhibition were measured after incubation for 48 h at 30 °C.

To determine the virus titer, MDCK cells were grown as a monolayer on 12‐well plates and then inoculated with 10‐fold serial dilution of wild‐type (WT) or mutant viruses in EMEM containing 1 μg·mL⁻¹ trypsin for 1 h at 37 °C. Cells were then overlaid with EMEM containing 0.6% UltrapureTM agarose (Invitrogen) and 1 μg·mL⁻¹ trypsin at 37 °C for 72–96 h. The infected cells were fixed in methanol and stained with 1% Crystal Violet (Sigma‐Aldrich, St. Louis, MO, USA). The plaques were visualized and counted.

The scanned animals (mice, Charles River, n = 3) were sacrificed at 10, 15 and 18 days of age in accordance with Schedule One of The Animals (Scientific Procedures) Act 1986 amendment regulations 2012. Within approximately 1 h euthanasia without any other preparation, they were placed in plastic cylinders of 1.9 cm in diameter (Fig. 1c). Any remaining empty space was filled by injecting gelatine (agarose gel 3 % approximately, UltraPure TM agarose from Invitrogen) into the cylinders, which solidified when cooling down and ensured that motion artefacts were minimised. Despite our best efforts to remove air pockets from the gelatine, some small air bubbles and a layer of air between the animals and the cylinder remained present. The relatively small effective vertical field of view of the Pixirad detector (approximately 2 cm) has restricted our scans to only a part of the animals. In line with previous studies in which the suitability of XPC micro-CT for preclinical imaging was investigated [12 (link), 13 (link), 15 (link)], we focussed on scanning the chest area (including the lungs). Although we were also interested in visualising the abdomen, this was not feasible due to air bubbles trapped in the intestinal tract. These moved and expanded during scans, causing strong motion artefacts which made an effective image reconstruction impossible.

The quality of RNA samples was evaluated by gel electrophoresis. A 1% UltraPure^TM Agarose (Invitrogen, Thermofisher Scientific, Harz, Germany) gel was prepared using 1X TAE buffer (Thermofisher scientific, Harz, Germany) dissolved in UltraPure^TM DEPC treated water (Invitrogen, Thermofisher Scientific, Harz, Germany) and supplemented with 0.5 μg/mL of ethidium bromide (VWR, Darmstadt, Germany). RNA samples and ladder were mixed with an equal volume of RNA loading dye (Thermofisher Scientific, Harz, Germany), incubated at 70 °C for 10 min, and cooled on ice. Subsequently, samples were loaded onto the gel and run for 45 min at 60 V. The eukaryotic 28 S and 18 S rRNAs and prokaryotic 23 S and 16 S rRNA were visualized with a UV transluminator (Gel Doc^TM XR, Bio-Rad, Feldkirchen, Germany).

RT-PCR and PCR products were analyzed by electrophoretic diffusion in 1.5% Ultrapure^TM Agarose (Invitrogen) gels submerged in 0.5X Tris-borate-Ethylenediamine Tetraacetic acid (EDTA). The size of DNA fragments was estimated by comparison with the 100 bp DNA Ladder (Invitrogen). A UV trans-illumination camera was used to visualize the DNA bands.

Yeast cultures were grown to logarithmic phase in rich YES medium to concentration 5 × 10⁶/mL, synchronized in 20 mM HU for 4 hours, subsequently released to fresh YES medium. At each time point 20 mL of cell culture was harvested, washed with cold 50 mM EDTA pH 8 and digested with litycase (Sigma, L4025) in CSE buffer (20 mM citrate/phosphate pH 5.6, 1.2 M sorbitol, 40 mM EDTA pH 8). Next cells were embedded into 1% UltraPure^TM Agarose (Invitrogen, 16500) and distributed into 5 identical agarose plugs for each time point. Plugs were then digested with Lysis Buffer 1, LB1 (50 mM Tris-HCl pH 7.5, 250 mM EDTA pH 8, 1 % SDS) for 1.5 hour in 55 °C and then transferred to Lysis Buffer 2, LB2 (1% N-lauryl sarcosine, 0.5 M EDTA pH 9.5, 0.5 mg/mL proteinase K) o/n at 55 °C. Next day LB2 was change for fresh one and digestion was continued o/n at 55 °C. After, plugs were kept at 4 °C. To visualize intact chromosomes one set of plugs was run on a Biorad CHEF-DR-III pulse field gel electrophoresis (PFGE) system for 60 h at 2.0 V/cm, angle 120°, 14 °C, 1800 s single switch time, pump speed 70 in 1x TAE buffer. Separated chromosomes were stained in ethidium bromide (10 μg/mL) for 30 min, washed briefly in 1x TAE and visualized with UV transilluminator.