Nanodrop nd 1000 spectrometer

The concentration of cfDNA was determined using a NanoDrop Spectrometer ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA) with a sample volume of 1 µL. Fragment distribution was assessed using the 4200 TapeStation device, using the High Sensitivity D5000 ScreenTape Assay with 1 µL sample, and 5 µL High Sensitivity D5000 Sample Buffer (Agilent, Santa Clara, CA, USA).

To obtain RNA, the cardiac tissues were homogenized in Qiazol lysis agent and with beads using Precellys lysing kit (Bertin Instruments, Montigny-le-Bretonneux, France) and purified using the RNeasy plus Universal Mini Kit (QIAGEN, Waltham, MA, USA). Reverse transcription was performed with 1 µg of total RNA using the High Capacity RNA to DNA kit (Thermo Fisher Scientific, MA, USA). The extracted RNA and cDNA concentration, respectively, were quantified using a NanoDrop Spectrometer ND-1000 (NanoDrop, Thermo Fisher Scientific Inc., Waltham, MA, USA).
The real-time quantitative PCR was performed on a 7900 HT Fast Real-Time PCR system (Thermo Fisher Scientific, MA, USA) using 1 µg cDNA, TaqMan® Gene Expression Assays, and 1 µL Gene Assay Mix for the genes HAS1-3, HYAL 1 and 2, CEMIP, CD44, VCAN, and TSG6 (Thermo Fisher Scientific, MA, USA). GAPDH (Thermo Fisher Scientific, MA, USA) was used as an endogenous reference gene. Forty cycles of amplification were performed. The gene of interest was normalized to the reference gene using the ΔCt method [24 (link)].

Undifferentiated iPSCs, iPSC-derived NPs or neurons at E and L stage of differentiation were rinsed with 1X DPBS followed by RNA extraction using RNeasy Mini kit (Qiagen) using the manufacturer’s protocol. RNA was quantitated using a Nanodrop Spectrometer ND-1000 (Thermo Scientific).

Genomic DNA was extracted from 25 mg of the renal cortex and 20 µL of urine of Leptospira-inoculated mice using QIAamp DNA Mini Kit (Qiagen, Victoria, Australia). The concentrations of extracted DNA from the renal cortex were determined using Nano Drop Spectrometer ND-1000 (Thermo Scientific, Rockford, IL, USA) and adjusted to 50 ng/µL with double distilled water (DDW) for quantitative PCR. DNA from urine samples was applied to quantitative PCR without concentration adjustment, as described by Soupé-Gilbert et al. [25 (link)].

Preoperative blood samples were usually collected on the day prior to RC at a median of 39 days [interquartile range (IQR): 27; 61] after the preceding TURB. Serum was prepared from 6 ml whole blood by 2 centrifugation steps of 3000 g and 16,000 g each for 10 min. Leukocytes (reference) were extracted from 6 ml EDTA blood supplemented up to 50 ml with lysis buffer containing 0.3 M sucrose, 10 mM Tris–HCl pH 7.5, 5 mM MgCl2 and 1% Triton X100 (Sigma, Taufkirchen, Germany). Following incubation for 15 min on ice, the isolation and purification of the leukocytes were carried out by 2 centrifugation steps at 2500 g, at 4 °C for 20 min. cfDNA was extracted from 2 ml serum using the PME free-circulating DNA Extraction kit (Analytik Jena), while DNA was extracted from leukocytes using the Qiamp DNA Blood Mini kit (Qiagen, Hilden, Germany). These DNA extractions were carried out according to the manufacturer´s instructions and similar to the procedure as described above. Quantification and quality of the extracted cfDNA were determined spectrophotometrically using the NanoDrop Spectrometer ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA).

Human stools were stored in a −20 °C refrigerator as soon as the stool was received (1–2 g) using stool paper and stool box and transferred to a −80 °C refrigerator in 1 week. Stool samples (1 g) were suspended in 10 volumes of Buffer ASL (Qiagen, Hilden, Germany) and homogenized by using a TissueLyser Ⅱ (Qiagen) with 5 mm stainless steel beads. Genomic DNA from stools was extracted by utilizing QIAamp DNA Stool Mini Kit (Qiagen). All measurements were performed according to the manufacturer’s instructions. The concentration and purity of each extracted DNA were determined using a NanoDrop spectrometer ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) and stored at −20 °C until further processing.

The dung beetles (12 females and 12 males) collected in 2013 and 2014 were surface sterilized and dissected to obtain their digestive tract. Each tissue sample was homogenized in lysis buffer (20 mM Tris-Cl, 2 mM sodium EDTA, 1.2% Triton X-100 and 20 mg/mL lysozyme at a pH of 8.0) with a 5 mm stainless steel bead using a TissueLyser (Qiagen). The DNA from the homogenates was isolated using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The prepared brood ball (n = 8) and cattle dung (n = 3) samples were homogenized with glass beads by vortexing and then centrifuged at 14,000 rpm for 1 min to pellet stool particles. The supernatants were used for DNA extraction with the use of the QIAamp DNA Stool Mini Kit (Qiagen). DNA extraction from the habitat soil samples (n = 2) was performed using the PowerMax^® Soil DNA Isolation Kit (MO BIO, Carlsbad, CA, USA) following the manufacturer’s protocol. The extracted DNA samples were purified using PowerClean DNA Clean-up kit (MO BIO) to eliminate PCR inhibitors. The final DNA extracts were checked for purity and concentration using a NanoDrop spectrometer ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) and stored at −20 °C until further processing.