Allprep powerfecal dna rna kit

The Qiagen Allprep PowerFecal DNA/RNA kit (Qiagen, Redwood City, CA, USA) was used for microbial DNA extraction. For each cat, the weight of fecal specimens was measured (Table 2) before being placed into a Microbial Lysis Tube for homogenization using a PowerLyzer24 instrument (Qiagen, Redwood City, CA, USA). DNA extraction procedures were conducted for all fecal samples in the same batch to minimize technical variability. The DNA concentrations were measured using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and the A260/A280 absorption ratios were determined with a NanoDrop One C Microvolume Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 500 ng of DNA from each sample was fragmented into 500-bp fragments using an M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). The WGS metagenomic libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA). TapeStation 4,200 (Agilent Technologies, Santa Clara, CA, USA) was utilized to evaluate the library size distributions. Subsequently, the final libraries were quantified using qPCR before being sequenced on an Illumina NovaSeq6000 sequencing platform in 150-bp paired-end mode by Novogene Corporation Inc. in Sacramento, CA, USA.

RNA was isolated using the AllPrep Power fecal DNA/RNA kit (Qiagen). For initial input, brushes in RNAlater were vortexed at maximum speed for 15 s, and 200 μL of this suspension was used. The manufacturer’s protocol was then followed with the exception of incubating samples for 15 min in the presence of lysis buffer and 25 μL of dithiothreitol (DTT) prior to removal of solid tissue for the biopsy samples, followed by homogenization of samples for both methods in the same manner. Quality control was performed using a Thermo Scientific NanoDrop 2000 spectrophotometer, ensuring 260/280-nm light ratios of >1.8 for all samples. Libraries were then constructed using 5 to 10 ng of RNA for each sample and using the Next Ultra II directional RNA library prep kit with rRNA depletion (New England Bioscience) in a paired-end fashion with 2 × 150-bp paired-end reads. Libraries underwent quality control via tape station prior to multiplexing at a concentration of 4 nM, and sequencing was performed on an Illumina MiSeq platform (San Diego, CA, USA) at the University of Colorado Genomics core with >6 Gbp of data output per sample. Actual sequencing depth was determined by FastQC and is presented in Table S1 in the supplemental material.

DNA from the fecal samples was isolated using Qiagen AllPrep^® PowerFecal^® DNA/RNA Kit according to the manufacturer’s instructions. A modification made was that fecal samples were lysed with provided lysis buffer using a vertical Disruptor Genie for 5 min at 3000 rpm. Fecal DNA samples were measured by Qubit quantification. PCR reactions were performed using the 16S primer for bacterial genomes and the ITS2 primer for fungal genomes. Fecal DNA was also used for qPCR and RT-qPCR to detect C. auris presence and viability. A standard curve was produced by performing a serial dilution of 10⁸ to 10¹ of the C. auris cells. These dilutions were then processed identically to the fecal samples collected to extract and purify DNA and RNA. A TaqMan qPCR reaction was run on the DNA samples using the ITS2 primer and probes for C. auris. The PerfeCTa Multiplex qPCR ToughMix was spiked with bicoid plasmid and respective primers after pipetting of the negative template control wells. Each sample and control were run in duplicate on a 7500 Fast instrument for 45 cycles. A standard curve was generated and used to calculate the relative concentrations of C. auris in the fecal pellets depending on their cycle threshold value. The fecal pellet DNA samples were run identically to the standard curve samples. 16S Ilumina sequencing was done as per the manufacturer’s instructions.

Two fecal samples were collected from each cat using a fecal loop with and without lubrication (N = 14 total; Supplementary Table S1) (32 (link)). At least 200 mg feces were used for DNA extraction by Qiagen Allprep PowerFecal DNA/RNA kit (Qiagen, MD), following the protocols provided by the manufacturer. During DNA extraction, fecal samples were homogenized by the Qiagen PowerLyzer24 instrument (Qiagen, MD) to achieve homogeneous results. The extracted DNA concentrations were measured by the Qubit 3 Fluorometer (Invitrogen, CA), and A260/A280 absorption ratios were assessed using the NanoDrop One C Microvolume Spectrophotometer (Thermo Fisher Scientific, MA). DNA fragmentation was performed by M220 Focused-ultrasonicator (Covaris, MA) on 1.5–2 μg of DNA for each sample to achieve fragmented DNA of ~500 bp. NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, MA) was used to construct WGS metagenomic sequencing libraries using the fragmented DNA. Final library concentrations and size distributions were measured by LabChip GX Touch HT Nucleic Acid Analyzer (PerkinElmer, MA) before being sequenced on an Illumina NovaSeq6000 sequencing machine on the 150-bp paired-end mode at the Genomics Service Laboratory at the HudsonAlpha Institute for Biotechnology (Huntsville, AL).

Genomic DNA and total RNA were extracted from the same flash‐frozen samples using Allprep Powerfecal DNA/RNA kit (Qiagen, Hilden Germany) following the manufacturer's protocol but an additional phenol–chloroform extraction step of 700 μl was performed after lysis. DNA yield was measured by using Qubit™ dsDNA HS Assay Kit (Qubit, Waltham, Massachusetts, USA), split into two aliquots for ribosomal 16S rRNA amplicon sequencing and metagenomic shotgun sequencing and was stored at −20°C. RNA yield was measured via Bioanalyzer (Agilent, Santa Clara, California, USA) with Pico and Nano chips depending on the sample concentration and stored at −80°C for further analysis.