Zr bashingbead lysis tube

Microbial DNA was isolated from all samples using the ZymoBIOMICS DNA Miniprep Kit, following the manufacturer’s instructions with some modifications; the cell pellet was re-suspended in 1,000 μL of DNA/RNA Shield (Zymo Research) and 250 μL of this cell suspension was added to 250 μL of lysis buffer in ZR BashingBead™ lysis tubes (Zymo Research) containing 0.1 mm and 0.5 mm beads. All samples underwent 5 rounds of bead beating using a MP FastPrep 24 (MP Bio, United States) for 20 s at 6.0 m/s with 1 min breaks between rounds. Following bead beating, samples were incubated at 70°C for 10 min to increase the effectiveness of DNA isolation. The remaining purification steps followed the manufacturers protocol, and all samples were eluted in 100 μL of distilled H₂O. DNA quantity and quality was assessed with a Nanodrop 2,100 spectrophotometer and visualization in a 1% agarose gel.

Yeast strains were grown on LB plates at 30 °C for 48 h. To isolate the DNA, the cell biomass was collected and extracted in ZR BashingBead Lysis Tubes (Zymo Research) containing 750 μl of lysis solution, according to the manufacturer’s instructions. Yeasts were taxonomically assigned based on the internal transcription spacers (ITS) ITS1-5.8S rRNA-ITS2, using the ITS1-ITS4 primer pair (White et al. 1990 ). In a final volume of 25 μl, the PCR reaction contained 2.5 mM MgCl₂, 0.3 mM dNTPs, 0.3 μM of each primer, 0.25 μl Taq polymerase (Thermo Scientific), 1 × PCR buffer, and 1 μl of the appropriate concentration of DNA. The amplification conditions were initial denaturation at 95 °C for 5 min, followed by 40 cycles of 1 min at 94 °C, 1 min at 55.5 °C and 2 min at 72 °C, and a final 10 min extension at 72 °C. The PCR reaction products were processed and sequenced as for 16S RNA gene amplification.

For miRNAs analysis, total RNA was extracted from the rat skeletal muscle samples using glass beads for rupture and Trizol™ Reagent. In brief, a fraction between 50 and 100 mg of frozen tissue was collected in a 2 mL tube with 2 mm glass beads (ZR BashingBead Lysis Tubes, Zymo Research) and 1 mL of Trizol™ Reagent. Then, 200 µl of clorophorm was added and mixed to be centrifuged at 12, 000 g for 15 min at 4 °C. After this step, total RNA extraction was performed according to manufacturer’s recommendations. The obtained RNA was quantified with an UV- spectrophotomer NanoDrop™ 2000 (Thermo Scientific), and the integrity was evaluated qualitatively in agarose gels.

This protocol was intended for total nucleic acids extraction using the PowerViral^® Environmental RNA/DNA Isolation Kit, which is a filter-based technique. The manufacturer’s instructions were followed with a slight modification. Briefly, 200 µL of the mock sample was mixed with 600 µL of prewarmed PV1 buffer and 6 µL of β-mercaptoethanol (Sigma, Heidelberg, Germany). The mixture was added to ZR BashingBead Lysis Tubes with mixed sizes (0.5 mm and 0.1 mm) of beads (Zymo Research, Irvine, USA). The tube was placed in a TissueLyser (Qiagen, Hilden, Germany) for 25 s, followed by a 5-s break and another 25 s of agitation at 30 Hz. The resulting mixture was centrifuged at 4 °C (5430 R; Eppendorf, Hamburg, Germany), and the supernatant was obtained by following the kit instructions. The eluted samples were labeled “MoBio”.

Sample collection was performed as described before (Ramqvist et al., 2011 (link)). However, both the sampling buffer and DNA extraction method were modified, the latter now performed as reported by Hugerth et al. (2018 (link)). Briefly, cervical swabs preserved in DNA/RNA Shield (Zymo Research Corp, Irvine, CA) where beads were beaten with ZR Bashing Bead Lysis Tubes (0.1 and 0.5 mm from Zymo Research Corp, Irvine, CA) at 1,600 rpm with the 96 FastPrep machine (MP Biomedicals, Santa Ana, CA, USA) for 1 min. The sample buffer was then spun at 4,400 rpm for 4 min to separate out the beads. Thereafter, the supernatant was incubated with lysozyme buffer (20 mM Tris-Cl, 2 mM sodium-EDTA, 100 g/ml lysozyme; Sigma, St. Louis, MO, USA) at 37°C for 3 h while shaking at 1,000 rpm. The DNA extraction was then conducted with the ZR-96 Genomic DNA MagPrep kit (Zymo Research Corp, Irvine, CA) according to the manufacturer's instruction. Finally, the extracted DNA was eluted from the magnetic beads with 70 μl Elution Buffer (10 mM Tris-Cl, pH 8.5; Qiagen, Venlo, Netherlands) and the purified DNA was stored at −20°C before HPV genotyping. For each extraction, DNA-free water samples were included as negative controls for potential contamination.

Stool samples were collected from the participants and frozen at −80 °C immediately after delivery. Sample transfer was performed on dry ice. Total genomic DNA was extracted from 120 mg fecal material using the ZR BashingBead lysis tubes (0.1 and 0.5 mm, Zymo Research, Freiburg, Germany) in combination with the chemagic DNA Stool Kit (Perkin Elmer, Rodgau, Germany) according to the manufacturer’s instructions [12 (link),13 (link),14 (link)]. A mechanical lysis step was added after the addition of the lysis buffer using the Precellys 24 Tissue Homogenizer (Bertin Instruments, Frankfurt am Main, Germany). After extraction, DNA was stored at −20 °C until further analysis.