Asl buffer

Total DNA from the microbial population was extracted from the probiotic product by weighing 0.1 g in a 2 mL tube containing 0.3 g of 0.1 mm zirconium beads and 700 µL of ASL buffer (Qiagen, Hilden, Germany). The mixture was homogenized, and the DNA released from the bacterial cells by 3 min of rigorous minibead beating in Qiagen/Retsch MM300 TissueLyser (Qiagen). Final DNA purification was done following the manufacturer’s protocol using QIAamp DNA Mini Kit (Qiagen) and stool buffer (Qiagen). The purity and concentration of the extracted DNA was checked using the SPECTROstar^Nano (BMG Labtech, Ortenberg, Germany) spectrophotometer.

For DNA extraction from fecal pellets, each frozen pellet was transferred to a 2 ml tube containing Lysing Matrix E beads (MP Biomedicals) and 1.4 ml ASL buffer (Qiagen), then homogenized using a FastPrep-24 5G benchtop homogenizer (MP Biomedicals). DNA was extracted after homogenization using the QIAamp DNA stool kit (Qiagen) according to manufacturer’s protocols. Oocyst numbers were quantified using qPCR with the C. parvum GAPDH primers as described above. To check for the successful insertion of the target sequence into either the tk or uprt locus, PCR was performed on 1 μl purified fecal DNA using Q5 Hot Start High-Fidelity 2X master mix (New England Biosciences) with primers listed in Table S2 at a final concentration of 500 nM each. PCR reactions were performed on a Veriti 96-well Thermal Cycler (Applied Biosystems) with the following cycling conditions: 98°C for 30 secs, followed by 35 cycles of 98°C for 15 secs, 64°C for 30 secs, and 72°C for 1.5 mins, with a final extension of 72°C for 2 mins. PCR products were run on 1.5% agarose gel containing GelRed (Biotium, diluted 1:10,000) and imaged on a ChemiDoc MP Imaging System (Bio-Rad).

Unflushed cecal tissue and content was suspended in 1.4 ml ASL buffer (Qiagen) and homogenized with 2.8 mm ceramic beads followed by 0.5 mm glass beads using an OMNI Bead Ruptor (OMNI International). DNA was extracted from the entire resulting suspension using QIAamp DNA Stool Mini Kit (Qiagen) according to manufacturer’s protocol. DNA was quantified using Qubit broad range DNA assay (Life Technologies). The V4 region of 16s rRNA gene was amplified using universal primers (515f and 806r) (Caporaso et al., 2012 (link)). Individual samples were barcoded, pooled to construct the sequencing library, and then sequenced using an Illumina Miseq (Illumina, San Diego, CA, United States) to generate pair-ended 250 nt reads. Quantitative PCR was performed for A. muciniphila as described in Schneeberger et al. (2015) (link) with DNA for standard curve isolated from the cultivated microbe.

Stool samples were collected by the participants at home and stored at room temperature until delivery to the clinic, where samples were stored at −80 °C. The maximum time between sampling and delivery to the clinic was 24 h. Total genomic DNA was isolated from 100–150 mg of faecal material using a modification of the IHMS DNA extraction protocol Q^{48 (link)}. Samples were extracted in Lysing Matrix E tubes (MP Biomedicals) containing ASL buffer (Qiagen), vortexed for 2 min and lysed by two cycles of heating at 90 °C for 10 min followed by two bursts of bead beating at 5.5 m s⁻¹ for 60 s in a FastPrep-24 Instrument (MP Biomedicals). After each bead-beating burst, samples were placed on ice for 5 min. Supernatants were collected after each cycle by centrifugation at 4 °C. Supernatants from the two centrifugations steps were pooled and a 600-µl aliquot from each sample was purified using the QIAamp DNA Mini kit (QIAGEN) in the QIAcube (QIAGEN) instrument using the procedure for human DNA analysis. Samples were eluted in 200 µl of AE buffer (10 mM Tris·Cl; 0.5 mM EDTA; pH 9.0). Libraries for shotgun metagenomic sequencing were prepared by a PCR-free method; library preparation and sequencing were performed at Novogene (China) on a NovaSeq instrument (Illumina) with 150-bp paired-end reads and at least 6G data per sample.

Total DNA was extracted from the 13 frozen fecal samples of HIV-infected patients using a modification of the Qiagen stool procedure and the QIAamp® DNA Stool Mini Kit (Qiagen, Courtaboeuf, France) [5 (link)]. Briefly, aliquots of 200 mg of feces were added to tubes containing a 200 mg mixture of 0.1, 0.5, and 2 mm zirconium beads and 1.5 ml of ASL buffer (Qiagen). The sample was bead-beaten at 3200 rpm for 90 seconds, followed by heating at 95°C for 10 minutes. The final pellet was suspended in 180 μl of tissue lysis buffer and incubated with proteinase K for 2 hours at 55°C. Then, DNA was prepared from the solution by using QIAamp spin columns (Qiagen) in an Eppendorf microcentrifuge, following the manufacturer’s instructions.

For DNA extraction, ASL buffer (QIAGEN GmbH, Hilden, Germany) was used, following the manufacturer’s instructions. Briefly, 500 µL of ASL buffer was added to 15 µL of the stool sample in Cary Blair medium (human samples) or a feces suspension in sterile distilled water (animal samples); the mix was vortexed for 20 s, incubated for 10 min at 95 °C, and then centrifuged at 13,000 for 5 min. The supernatant was used for DNA extraction using the EZ1 automated extraction system (QIAGEN).

DNA was extracted by using the QIAmp DNA mini-kit (Qiagen, Valencia, CA, United States). 50 mg of the cecum were mixed with 0.3 g of 0.1 mm sterile zirconia beads with 700 μL of ASL Buffer (Qiagen) in a 2 mL bead beating tube and homogenized for 3 min in the Mini-Beadbeater-16. The following DNA extraction procedures were performed according to the QIAmp DNA Mini Kit manufacturer’s instructions. Microbiota analysis of the supplementary model was performed by qRT-PCR and using the specific primers suggested by Bergström et al. (2012) (link).