DNA extraction, amplification, and sequencing from mice feces

Total DNA was extracted from fecal samples using the PowerSoil DNA Isolation Kit (MoBio, Carlsbad, USA), according to the manufacturer’s protocol, and following a 2-min bead-beating step (Biospec). The V4 region of the bacterial 16S rRNA gene was amplified using the 515F and 806R barcoded primers following the Earth Microbiome Project protocol [10]. PCR protocol included 2 μl 515F primer (10 μM), 2 μl 806R primer (10 μM), 25 μl PrimeSTAR Max PCR Readymix (Takara, Mountain View, USA), 17 μl ultra-pure water, and approximately 20 ng of DNA. PCR reaction conditions included 3 min at 95 °C followed by 30 cycles of [10 s at 98 °C, 5 s at 55 °C, and 20 s at 72 °C], and final elongation for 1 min at 72 °C. Amplicons were purified using AMPure XP magnetic beads (Beckman Coulter, FL, USA) according to the manufacturer’s protocol. DNA was quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, USA), and equimolar amounts of DNA were pooled from each sample, to ensure equal read depth. After running on a 2% agarose E-Gel (Invitrogen, Carlsbad, USA), DNA was extracted from the gel using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and sequenced using the Illumina Miseq platform at the Genomic Center, Azrieli Faculty of Medicine, BIU, Israel.