Dneasy powersoil pro kit

Two weeks after sample storage, DNA from all aliquots was extracted using the DNeasy PowerSoil Pro Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s guidelines. An additional heating step was included, where all samples were heated at 65 °C for 10 min prior to bead beating (Bullet blender storm, 3 min at speed 8). As per the manufacturer’s recommendations, stool in the ‘SSK ambient’ tube was aliquoted immediately prior to DNA extraction. The first aliquot of 250 µL was taken without re-homogenising the kit; however, the second 250 µL aliquot was taken after re-homogensation, as per the recommendations (Figure 1). All extracted DNA was then cleaned prior to analysis using the GeneJET PCR Purification Kit (ThermoFisher Scientific, Waltham, MA, USA) as per the manufacturer’s instructions.

Gut microbiome samples were collected by isolating 1–2 individual fecal pellets from mice of interest. These pellets were stored in individually wrapped, sterile Eppendorf Safe-Lock tubes (Eppendorf 022600044) at −80°C until DNA extraction. Genomic DNA was extracted from fecal samples using a Qiagen DNeasy PowerSoil Pro kit as described by the manufacturer’s instructions (Qiagen 47014).

The DNA extraction procedure has been described by (34 (link)). Briefly, DNA was extracted from 300 mg of digesta (nine samples/group), 200 µL of mucosal bacterial suspension (six samples/group), and 200 mg of each feed (three aliquots/feed). The DNeasy PowerSoil^® Pro Kit (Qiagen, Milan, Italy) was used for extraction according to the manufacturer’s instructions. The concentration and purity of DNA were spectrophotometrically measured using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, Milan, Italy). Bacterial DNA was stored at -20°C until NGS library preparation.

As already described in Gazzola et al. (2022) (link), anaerobic digestate samples were collected at different sampling times to perform the analysis of microbial community composition over the reactor operation. A small aliquot (2 mL) was centrifuged at 15,000 rpm for 2 min, the resulting pellet was immediately stored at −20°C and used for DNA extraction with DNeasy PowerSoil Pro Kit (QIAGEN - Germantown, MD) following the manufacturer’s instructions. After checked the quality (1.6 < A260/280 < 1.8 and A260/230 > 2) with a Nanodrop 3,300 (Thermo Scientific, Italy), the genomic DNA was used for the high-throughput sequencing of 16S rRNA gene. The V1-V3 region of bacterial 16S rRNA gene (27F 5′- AGA​GTT​TGA​TCC​TGG​CTC​AG-3; 534R 5-ATT​ACC​GCG​GCT​GCT​GG-3) was sequenced following library preparation and protocol described in Crognale et al. (2019) (link). The samples were paired end sequenced (2 × 301bp) on a MiSeq platform (Illumina) using a MiSeq Reagent kit v3, 600 cycles (Illumina, United States) following the standard guidelines for preparing and loading samples, with 20% of Phix control library. Bioinformatic analysis were performed using QIIME2 v. 2018.2 (Bolyen et al., 2019 (link)) as described in Crognale et al. (2021) (link). The Dataset is available through the Sequence Read Archive (SRA) under accession PRJNA1052454.

Bacterial DNA was extracted from 0.25 g of the soil using a DNeasy PowerSoil Pro Kit (Qiagen) according to the protocol. The concentration of the isolated DNA was measured with a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and each of the samples was diluted to a final concentration of 10 ng/µL using DNase-RNase free water.

Seven- to 11-week-old male mice (C57BL/6 mEcad E16P KI^{77 (link)}) were infected intravenously in the tail vein as previously described^{77 (link)}. Fraternities were kept together in a cage during the whole experiment, except when separated to exclude that carriage was due to coprophagy. To quantify carriage at 30-days post-inoculation, faeces were collected from each individual mouse and weighted before being homogenised in 2 ml of PBS. CFU count was performed by serial dilution of homogenised faeces on ALOA plates (Biomérieux) as described in ref. ⁸ . Separate faecal pellets were collected pre-inoculation and/or 30-days post-inoculation and stored at −20 °C for DNA extraction for 16S rRNA gene sequencing. DNA was extracted with DNeasy PowerSoil Pro Kit (Qiagen).
For experiments with antibiotic treatment, 5-week-old female mice (C57BL/6 mEcad E16P KI^{77 (link)}) received orally every 12 h during 4 days PBS (as control) or the following antibiotics (similarly to^{57 (link)}): ampicillin (100 mg/kg, Sigma-Aldrich A9618), vancomycin (50 mg/kg, Sigma-Aldrich 75423), metronidazole (100 mg/kg, Abcam ab141218), neomycin (100 mg/kg, Gibco 11811-031), amphotericin B (1 mg/kg, ApexBio Technology B1885). The mice were inoculated, as described above, 4 weeks after the antibiotic treatment.

Samples were thawed on ice and DNA was extracted using the DNeasy PowerSoil Pro kit (Qiagen) and the TissueLyser LT (Qiagen) for sample disruption. DNA was normalized and the V4 hypervariable region of the bacterial 16 S rRNA gene was amplified in a PCR reaction (PCR1). Primers included sequences for sequencing adapters and barcodes suitable for the Illumina MiSeq instrument. Amplicons were diluted 1 in 5 and then underwent a second PCR amplification (PCR2) in which dual indexes were attached for downstream sample identification. PCR1 heated samples to 95 °C for 3 min then comprised 20 cycles of 95 °C for 45 s, 50 °C for 60 seconds and 72 °C for 90 seconds, followed by a 72 °C hold for 10 min. PCR2 heated samples to 95 °C for 3 min then comprised 25 cycles of 95 °C for 45 seconds, 55 °C for 60 seconds and 72 °C for 90 seconds, followed by a 72 °C hold for 10 min. Amplicons were pooled and purified using 0.8x NGS beads (NucleoMag), then the library pool was profiled using the Agilent TapeStation prior to sequencing on the MiSeq instrument (2x 250 bp paired-end sequencing).