Dneasy powersoil pro dna isolation kit

To disperse the bacterial entrapment, the collection tubes were vortexed slowly. The DNeasy PowerSoil Pro DNA Isolation Kit (Qiagen, Valencia, CA), catalogue number 47016–250, was used to efficiently extract genomic DNA from the swabs following the manufacturer’s instructions.

The remaining cell suspension (9 ml) was centrifuged in a swing-out rotor (Eppendorf A-4-62 Swing Bucket Rotor) at 4000 × g for 7 min to harvest the cells. The supernatant was discarded and cell pellets were stored at –80 °C. DNA was subsequently extracted from thawed cell pellets using a DNeasy PowerSoil Pro DNA Isolation Kit (Qiagen, as per instructions by the supplier). Final yields were quantified using a Qubit dsDNA BR Assay Kit (Invitrogen), and the purified DNA solution was stored at –20 °C until library construction. Each sample (10 ng DNA input) was then used to amplify the V3–V4 variable region of the 16 S rRNA gene, following the protocol by Illumina (16S Metagenomic Sequencing Library Protocol, https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223b.pdf).
Samples were indexed by using the Nextera XT Index kit (v2, sets A and B, Illumina) after which the DNA was again purified, pooled, and sequenced using a MiSeq v3 paired-end protocol (Lausanne Genomic Technologies Facility). Raw reads were analyzed using the Qiime2 platform on UNIX (version 2021.8)^{60 (link)}, and amplicon sequence variants (ASVs) were attributed to known taxa at 99% identity (operational taxonomic units, OTU) by comparison to the SILVA database (version 132).

Mouse faeces were snap-frozen in dry ice once they were collected from mouse intestines under aseptic conditions. All samples were kept at − 80 °C before DNA extraction. Bacterial genomic DNA was extracted using a DNeasy PowerSoil Pro DNA Isolation Kit (QIAGEN Ltd., Manchester, UK), as described previously [26 (link)] and the Human Microbiota Project [68 (link)]. All DNA samples were kept at – 80 °C before being sent for library preparation and sequencing at the Oxford Genomics Centre (Wellcome Centre for Human Genetics, University of Oxford, UK).