Quanti it pico green dsdna assay

Three replicate sub-samples were obtained from each soil sample under strict control conditions to avoid external contamination, as described by Rosa et al. [11 (link)]. Total DNA was extracted from these using the FastDNA Spin Kit for Soil (MPBIO, Solon, OH, USA). DNA quality was analyzed using agarose gel electrophoresis (1% agarose in 1× Trisborate-EDTA) and then quantified using the Quanti-iT™ Pico Green dsDNA Assay (Invitrogen). The internal transcribed spacer 2 (ITS2) of the rDNA was used as a DNA barcode for fungal identification [14 (link),15 (link)], using the primers ITS3 and ITS4 [16 ]. The Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2 was used for library construction and DNA amplification followed the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Part #15044223, Rev. B). Paired-end sequencing (2 × 300 bp) was performed on a MiSeq platform (Illumina) by Macrogen Inc. (Seoul, South Korea).

DNA was extracted from the Pakistani and Finnish participants’ fecal samples using the same procedure, Repeated Bead Beating (RBB) method,^{73 (link)} with the following modifications for automated DNA purification: Approximately, 0.25 g of fecal samples and 340 µL and 145 µL of lysis buffer were used on the first and second rounds of bead beating, respectively. Then, 200 µL of the clarified supernatant collected from the two bead beating rounds was used for DNA extraction with the Ambion Magmax™ −96 DNA Multi-Sample Kit (4413022, Thermo Fisher Scientific, USA) using the KingFisherTM Flex automated purification system (ThermoFisher Scientific, USA). DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA). Library preparation and Illumina MiSeq sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene using primers 341 F/785 R were performed as previously described.^{74 (link)} To characterize the gut fungal community, we performed ITS-1 sequencing for the Pakistani samples using a two-step PCR protocol described in detail elsewhere.^{75 (link)} PCR-amplicons of the ITS-1 region were generated using ITS1F and ITS2 primers.^{75 (link),76 (link)}

Fecal samples were collected at home at baseline and on the 2nd last day of each intervention arm. The samples were immediately stored at −20 °C and transported to the study centre for DNA extraction within 6 months. Bacterial DNA was extracted from approx. 125 mg of fecal matter using the Repeated Bead Beating (RBB) method [16 (link)] with the following modifications for automated DNA purification: After the beat beating steps, 400 μl of the cell lysate pooled from the two beat beating rounds was purified with the RSC Blood DNA kit AS1400 in a Promega Maxwell RCS instrument (Promega, Madison, WI, USA). DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA).

All eight bacterial strains that make up the VSL#3 product were individually provided by the manufacturer (courtesy of Actial Farmaceutical SRL, Rome, Italy), coded and cultivated as listed in S1 Table. Five ml of overnight-grown cultures were then further used for genomic DNA isolation. Cells were lyzed using lysozyme (20mg/ml), mutanolysin (10U/ml), 1% w/v SDS, 50 μg/ml RNase and 300 μg/ml proteinase K followed by an incubation of 30 min at 37 ^oC. Genomic DNA was then isolated from cell lysates with a RSC Blood DNA kit AS1400 according to the manufacturer’s protocol on a Promega Maxwell RCS instrument (Promega, Madison, WI, USA). DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA).

Bacterial DNA was extracted from the swabs using a previously described bead beating method^{25 (link)} with the following modifications: The swabs were vortexed in 0.5 ml of sterile ice-cold PBS, of which 175 μL was combined with 235 μL of RBB lysis buffer (500 mM NaCl, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 4% SDS) in a bead beating tube. The samples were bead beaten using a FastPrep-24 instrument at 5.5 m/s (MP Biomedicals, Inc., USA) with 0.1 mm zirconium-silica beads (Biospec Products, Bartlesville, OK, USA) for 1 min. Samples were then heated at +95 °C for 15 min with shaking 400 rpm and centrifuged at room temperature for 5 min at 13 000 rpm. The supernatant (200 μL) was used for DNA extraction with KingFisher Flex automated purification system (ThermoFisher Scientific, USA) and Ambion Magma Total Nucleic Acid Isolation Kit (Life Technologies, USA) using MagMAX Pathogen High Vol Duo program. DNA was quantified using Quanti-iT Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA). An aliquot of the DNA extract was sent to Karolinska Institutet, Sweden for Human papillomavirus (HPV) genotyping^{49 (link)}.

Fecal samples for the patients and controls were collected at home and immediately stored at -20°C. They were transported to a study center within 1 week. An uninterrupted frozen cold chain was ensured in the provision and handling of the fecal samples. Bacterial DNA was extracted from ca. 250 mg of fecal matter using the Repeated Bead Beating (RBB) method (13 (link)) with the following modifications for automated DNA purification: 340 μl and 145 μl of lysis buffer was added to first and second round of bead beating, respectively. 200 µl of the clarified supernatant collected from the two bead beating rounds was used for DNA extraction with the Ambion Magmax™ -96 DNA Multi-Sample Kit (Thermo Fisher Scientific, USA) using the KingFisherTM Flex automated purification system (Thermo Fisher Scientific, USA). DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA).
Blood samples were collected in the morning between 7 and 10 am after an 8- to 12-hour fast before taking morning medications. Sera were isolated with a standard protocol and stored at -80°C until analyses.