Ambion magmax total nucleic acid isolation kit

Fecal samples were collected by the participants at home and frozen immediately. They were transported to the laboratory frozen and stored at − 80 °C until processing.
Bacterial DNA was extracted using a previously described repeated bead-beating method^{35 (link)} with the following modifications for automated DNA purification: ca. 125 mg of fecal material were suspended in 1 ml of sterile ice-cold PBS, and 175 μl of fecal suspension 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 from the Ambion Magmax™ Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA). After repeated bead-beating, 200 μl of the supernatant was used for DNA extraction with a KingFisher™ Flex automated purification system (ThermoFisher Scientific, Waltham, MA, USA) using a MagMAX™ Pathogen High Vol. Duo program. DNA was quantified using a Quanti-iT™ Pico Green dsDNA assay (Invitrogen, San Diego, CA, USA) and 1 ng was used for V3–V4-region amplicon PCR of the 16S rRNA gene as previously described^{36 (link)}. Sequencing was carried out with Illumina HiSeq 2500 equipment in Rapid Run mode.

DNA was extracted from the stool samples using a bead-beating method.²⁰ (link) In short, ca. 250 or 340 mg of faecal material was suspended in 0.5 or 1 ml of sterile ice-cold PBS, and 250 μl of faecal suspension was combined with 340 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 from the Ambion MagMAX™ Total Nucleic Acid Isolation Kit (Life Technologies). After repeated bead-beating, 200 μl of the supernatant was used for DNA extraction with a KingFisherTM Flex automated purification system (ThermoFisher Scientific) using a MagMAXTM Pathogen High Vol. DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay (Invitrogen). The V3–V4 region primers (primers 341F/785R) of the 16S rRNA gene was amplification was performed according to the Illumina manual with slight modification for TrusSeq-tailed 1-step amplification.²⁵ (link) For infant samples until 12 months, 5 ng of DNA template was used for index-PCR (27–40 cycles) and 1 ng from thereafter and for adult samples. The library preparation protocol has been described in more detail previously.²⁶ (link) Sequencing was performed with Illumina MiSeq and HiSeq sequencing technology in the Functional Genomics Unit and Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland.