Cell lysis solution

Acetaldehyde and [ethyl-D₅]EtNH₃Cl were purchased from Millipore Sigma (St. Louis, MO, USA). In addition, 6-Chloropurine-2′-deoxyriboside was obtained from Carbosynth (Compton, UK). Water (LC-MS grade), methanol (MeOH, LC-MS grade), acetonitrile (ACN, LC-MS grade), 2-propanol (IPA, LC-MS grade), and formic acid (FA, 98% v/v) were purchased from Fisher Scientific (Hanover Park, IL, USA). Distilled water was purified by a Milli-Q system (Milford, MA, USA). Deoxyribonuclease I recombinant expressed by Pichia pastoris (R-DNase, 10,000 U/mg, phosphodiesterase-1 extracted from Crotalus adamanteus (PDE-1, 0.4 U/mg, recombinant alkaline phosphatase expressed by Pichia pastoris (R-ALP, 7000 U/mg, calf thymus DNA (CT-DNA, 5 mg), NaBH₃CN, Acetaldehyde, Tris base, double-filtration membrane Amicon Ultra (30 kDa cutoff, 0.5 mL), and single-filtration membrane Microcone (10 kDa cutoff, 0.5 mL) were purchased from Millipore Sigma (St. Louis, MO, USA). Silanized vials (0.3 mL, 1.2 mL, 4 mL, 20 mL) were purchased from ChromTech (Apple Valley, MN, USA). Cell lysis solution, protein precipitation solution, RNase A, and proteinase K were obtained from Qiagen (Hilden, Germany).

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.

Sorted T cells were pelleted and resuspended in cell lysis solution (Qiagen) and DNA was isolated from cell lysate using the Gentra Puregene kit (Qiagen) for 9 donors (D383, D466, D324, D299, D255, D233, HD1, HD2, and HD3). For 3 donors (D287, D280, and D229), DNA and RNA were extracted using an RNA/DNA kit (AllPrep DNA/RNA mini kit, Qiagen). Upon extraction of DNA from purified T cells, DNA was divided into equal parts for replicate amplification and sequencing. The amount of DNA sequenced per sample was constant for the first two replicates of each individual donor, with the exception of blood from D383, in which fewer cells were obtained. The quantity of DNA isolated and sequenced per sample is indicated in Additional File 1: Table S1.

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.