Powerbead pro

To assess the fecal microbiota, all dog owners were provided with two identical Nom Nom Plus Microbiome Testing Kits (Nashville, TN, USA), and instructed to use one kit to provide a stool sample at baseline (day 0) and the second kit to provide a stool sample at the end of the study (week 10). These collection kits have been previously utilized in other research studies [53 (link),71 (link),73 (link)]. Stool samples from both baseline and week 10 were received and processed in a single batch by Diversigen Inc. (New Brighton, MN, USA). DNA extraction and library construction protocols were performed as previously described [53 (link),78 (link)], with the exception that DNA was extracted using the Zymogen Quick-DNA Fecal/Soil Microbe 96 Mag Bead kit (Zymo Research, Irvine, CA, USA) using Powerbead Pro (Qiagen, Redwood City, CA, USA) plates with 0.5 and 0.1 mm ceramic beads. Extraction controls included a no template control (water) and a characterized homogenized stool. All samples were quantified with Quant-iT Picogreen dsDNA Assay (Invitrogen, Carlsbad, CA, USA). Subsequently, DNA amplification and library construction were performed with the Nextera XT DNA Library Preparation Kit (Illumina Inc., Foster City, CA, USA).

For bacterial DNA extraction 700μL of SL1 lysis buffer (NucleoSpin Soil kit, Macherey-Nagel) was added to the stool samples and tubes were heated at 95°C for 2h to inactivate SARS-CoV-2. Samples were then homogenized using the FastPrep-24TM instrument (MP Biomedicals) and extraction was pursued using the NucleoSpin Soil kit according to the manufacturer’s instructions. DNA concentration was assessed using a NanoDrop spectrophotometer. Samples with too low DNA concentration were excluded. DNA from human samples was extracted with PowerSoil Pro (Qiagen) on the QiaCube HT (Qiagen), using Powerbead Pro (Qiagen) plates with 0.5mm and 0.1mm ceramic beads. For mouse samples, the variable region 4 (V4) of the 16S rRNA gene was amplified by PCR using primers containing adapters for MiSeq sequencing and single-index barcodes. All PCR products were analyzed with the Agilent TapeStation for quality control and then pooled equimolar and sequenced directly in the Illumina MiSeq platform using the 2×250 bp protocol. Human samples were prepared with a protocol derived from ^{44 (link)}, using KAPA HiFi Polymerase to amplify the V4 region of the 16S rRNA gene. Libraries were sequenced on an Illumina MiSeq using paired-end 2×250 reads and the MiSeq Reagent Kitv2.