Protein precipitation solution

To isolate genomic DNA from the 279 water samples (93 days in triplicates) we used a bead beating approach. Filters were sterilely cut and placed into 2 mL screw-cap tubes, followed by the addition of 0.25 g of sterile 0.1 mm zirconium beads (BioSpec Products Inc., Bartlesville, OK). Cells were lysed by adding 750 μL of Cell Lysis Solution (Qiagen, USA) and shaken in a Mini Beadbeater-1 (BioSpec Products, Inc., Bartlesville, OK) at 5000 rpm for 60 s, followed by incubation at 80 °C for 5 min. Once samples were cooled down to room temperature, RNA was digested by adding 4 µL RNAse A (4 mg/mL), mixed by inverting the tubes, and incubated at 37 °C for 30 min. DNA was purified by adding 250 µL of Protein Precipitation Solution (Qiagen, USA), mixed by vortexing for 20 s, incubated on ice for 5 min and centrifuged at 13,000 rpm for 5 min. Supernatant was transferred to a new tube and centrifuged again to ensure removal of all precipitates. Subsequently, 750 µL of isopropanol were added to 750 µL of supernatant to precipitate DNA, and incubated at −20 °C overnight. DNA was recovered by centrifugation at 13,000 rpm for 5 min, and washed with 700 µL of 70% ethanol. Finally, the DNA was dried and resuspended in 100 µL of DNA hydration solution (Qiagen, USA).

Cell Lysis Solution (Qiagen, Hilden, Germany) and Protein Precipitation Solution (Qiagen) were used according to the manufacturer’s instructions to isolated genomic DNA from RCS-iPSCs. The information of primer pairs used to analyze PAX2 mutations is provided in Supplementary Table S4.

Total DNA extractions were performed by pelleting 1 mL of stationary phase cells. Pellets were resuspended in 600 μL Cell Lysis Solution (Qiagen) and lysed by incubation at 80°C for 5 min. A total of 50 μg of RNAse A was then added to the cell lysate and incubated at 37°C for 30 min to digest cellular RNAs. Proteins were precipitated from the lysate by adding 200 μL of Protein Precipitation Solution (Qiagen), vortexing and setting the sample on ice for 30 min, and pelleting for 10 min at 14,000 rpm. The supernatant was then mixed with 600 μL of isopropanol and inverted to precipitate the DNA. The DNA was pelleted via centrifugation at 14,000 rpm for 3 min, washed once with 70% ethanol, and resuspended in 100 μL of H₂O.

DNA extraction from the spleens was performed using ProteinaseK, RNaseA, CellLysis Solution, Protein Precipitation Solution, and DNA Hydration Solution from Qiagen according to the manufacturer’s manual (Qiagen, Venlo,the Netherlands). In-solution targeted enrichment of exonic sequences from both the phenotypically mutant and the control wild-type littermate mouse using the SureSelectXT Mouse All Exon kit (Agilent, Santa Clara, CA, USA) was performed. Libraries were sequenced as 100 bp paired-end runs on a HiSeq 2000 system (Illumina, San Diego, CA, USA). Read alignment to mouse genome assembly mm9 was done with Burrows-Wheeler Aligner (BWA, version 0.5.9), and a total of 10 and 9.2 Gb of mapped sequence data corresponding to an average coverage of 120× (>95 % of the target being covered >20×) and 113× for the mutant and control, respectively, were yielded. Single-nucleotide variants (SNVs) and small insertions and deletions (indels) were detected with SAMtools (v. 0.1.7).

Genomic DNA was isolated from all four infected calves and used for PCR. Briefly, tfBbo5480 and WT B. bovis short-term in vitro cultures prepared as above were expanded to 5% parasitemia. The erythrocytes were pelleted and washed with phosphate-buffered saline (PBS) (Thermo Fisher Scientific). Erythrocytes were lysed using red blood cell lysis solution (Qiagen) and incubated for 5 min at room temperature. Lysed red cells were centrifuged for 3 min at 2800× g and the supernatant was discarded. Parasite pellets were suspended in 40 µL of PBS and 500 µL of cell lysis solution (Qiagen) with 20 μg/mL of Proteinase K (Qiagen) and incubated at 56 °C for 30 min. Proteins were removed using Protein Precipitation Solution (Qiagen), and DNA precipitated using isopropanol, washed with 70% ethanol, and suspended in 50 μL of DNA hydration solution (Qiagen). PCR was performed from gDNA samples using primer sets (Table 1). PCR cycling conditions consisted of 95 °C for 3 min followed by 39 cycles of 95 °C for 30 s, 55 °C for 40 s, and 68 °C for 3.5 min, with a final extension of 72 °C for 5 min. PCR products were visualized by 1% agarose gel electrophoresis. PCR amplicons were cloned into PCR 2.1-TOPO^® (Thermo Fisher Scientific) and submitted for sequencing (Eurofins MWG Operon).