Fastprep fp100a

Fecal samples (20 mg) were suspended in 450 µl extraction buffer (100 mM Tris/HCl, 40 mM EDTA, pH 9.0), and 50 µl 10% SDS. Glass beads (300 mg, 0.1 mm diameter) and 500 µl buffer-saturated phenol were added to the suspension, and the mixture was vortexed vigorously for 30 s using a FastPrep FP 100A (MP Biomedicals, LLC, Santa Ana, CA, USA) at a power level of 5. After centrifugation at 14,000× g for 5 min, 400 µl of the supernatant was extracted with phenol/chloroform, and 250 µl of supernatant was precipitated with propan-2-ol. Purified DNA was rinsed with 300 µl 70% ethanol, and then suspended in 200 µl Tris/EDTA buffer (pH 8.0).

DNA was extracted from stool samples as previously described [15 (link)]. Stool samples were resuspended in a buffer containing 4 M guanidium thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA and mixed with zirconia beads using the FastPrep FP100A instrument (MP Biomedicals, Irvine, CA, USA). DNA was extracted using a Magtration System 12GC and GC series MagDEA DNA 200 reaction cartridge (Precision System Science, Tokyo, Japan). The final concentration of the DNA sample was adjusted to 10 ng/μl.

Frozen fecal samples were thawed on ice, 100 mg of each sample was suspended in 4 M guanidium thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA, and the samples were then beaten with zirconia beads using a FastPrep FP100A instrument (MP Biomedicals, USA). DNA was extracted from the bead-treated suspensions using a Magtration System 12GC and GC series MagDEA DNA 200 (Precision System Science, Japan). DNA concentrations were estimated by spectrophotometry using an ND-1000 instrument (NanDrop Technologies, USA), and the final concentration of the DNA sample was adjusted to 10 ng/µL.

DNA and RNA were extracted using AllPrep DNA/RNA Micro kit (Qiagen), including a bead beating step to ensure lysis of microbial cells. Briefly, the tissue samples stored in RNAlater were thawed at 37°C, pelleted by adding equal volume of PBS and spinning at 5,000 rpm for 5 minutes, and resuspended in 600 μL of RLT plus lysis solution. The lysate was transferred into DNase/RNase free tubes containing 200 mg 100-μm zirconium beads (Molecular biology grade, PFMB 100-100-12, OPS Diagnostics), and bead beaten at 6 m/s for 1 minute at room temperature using FastPrep FP100A instrument (MP Biomedicals). The lysate was used to sequentially isolate DNA and RNA as per manufacturer's instructions. For RNA, in-column DNase treatment was done using RNase-Free DNase Set (Qiagen). Aliquots of RNAlater were used as extraction negative control. The purity of RNA and DNA was assessed by measuring 260/280 ratio using Nanodrop (Thermo Fisher Scientific) and quantity was measured using Qubit RNA HS Assay Kit and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), respectively. The RNA integrity (RIN) and size distribution was assessed using Agilent RNA 6000 Pico Kit on Bioanalyzer 2100 (Agilent Technologies). DNA and RNA concentrations and RIN numbers for the samples are presented in Supplementary Data S1.

Fungal strains were cultured on Sabouraud dextrose agar. Genomic DNA was extracted from overnight cultures of A. fumigatus mycelia by the urea-phenol method. Mycelia were mixed with 0.5 mm size glass beads, 0.5 ml of PCI (phenol/chloroform/Isoamyl alcohol) solution and 0.5 ml DNA extraction buffer (50 mM Tris–HCl, pH 8.0, 20 mM EDTA, 0.3 M NaCl, 0.5% SDS, 5 M urea), and disrupted by Fast Prep FP100A (MP-Biomedicals, Santa Ana, USA) for 3 cycles of 30 s each at a speed of 4.0 m/s. After centrifugation, the upper layer was transferred to a new tube and subjected to ethanol precipitation. The resulting DNA pellet was suspended in 100 μL TE buffer. DNA concentration was determined by the methods described in our previous paper^{22 (link)}.
All A. fumigatus strains were submitted to antifungal susceptibility tests according to the CLSI M38 protocol (https://clsi.org/standards/products/microbiology/documents/m38/), using Eiken Dried Plates (9DEF47, Eiken Chemical Co., Tokyo, Japan).