Qiaamp powerfecal dna isolation kit

The mouse intestines were immediately transported to the laboratory at 4 °C, where they were stored in a deep freezer at −70 °C until further analysis. Genomic DNA was extracted using a QIAamp Power Fecal DNA Isolation Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The concentration and purity of the extracted genomic DNA were confirmed by agarose gel electrophoresis and UV-visible spectrophotometry (Mecasys Co., Ltd., Daejeon, Republic of Korea). PCR amplification of 16S rRNA was performed using primers for the V3–V4 hypervariable region. The PCR conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, and final extension at 72 °C for 1 min 30 s. The amplicons were purified using a NucleoSpin Clean-up Kit (Macherey-Nagel, Dueren, Germany) following the manufacturer’s instructions, and then used to construct sequencing libraries with an Illumina TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA, USA). For each sample, barcoded V3–V4 PCR amplicons were sequenced using the Illumina Miseq platform (MiSeq Reagent Kit v2, Illumina, CA, USA). Finally, amplification and sequencing of the V3–V4 region of the 16S rRNA gene was performed by ChunLab, Inc. (Seoul, Republic of Korea).

DNA was extracted using the QIAamp PowerFecal DNA Isolation Kit (Qiagen, Germany) according to the manufacturer’s instructions. Samples that did not meet the purity requirements of the sequencing provider were subjected to ethanol-based purification. The extracted DNA was frozen at − 20 °C until delivery on ice to the Centre for Proteomic and Genomic Research (CPGR) where 16S rRNA gene amplicon sequencing was performed on the Illumina MiSeq platform. Previously published primers^{16 (link)} were used to target the V4 hypervariable region. The MiSeq Reagent v3 Kit (600 cycles) was used to generate sequencing libraries, which were spiked with 10% of a 5 pM PhiX sequencing control. The ZymoBIOMICS Microbial Community DNA standard (Zymo Research, USA) and batched negative extraction controls were included in the sequencing run, which was set to produce 2 × 200 bp paired-end reads. The DNA purity requirements and sequencing controls are described in detail in the “Supplementary material”.

Whole genomic DNA was extracted from fresh fecal samples using the QIAamp PowerFecal DNA Isolation kit (Qiagen, Hilden, Germany), as per the manufacturer’s protocols. This method has been shown to yield sufficient concentration and quality of DNA for use in 16S rRNA amplicon analysis [32 (link)]. For logistical reasons, 26 fecal samples (18 samples from the vegan group and 8 samples from omnivore group) were pooled to create a total of 5 vegan and 5 omnivore composite samples. DNA samples were submitted to the University of Minnesota Genomics Center (UMGC) for 16S rRNA sequencing. Pipeline analysis and associated primers, reagents, and equipment were conducted using previously described methods to reduce potential source library bias [33 (link)]. Primers used were 784F (5′-RGGATTAGATACCC-3′) and 1064R (5′-CGACRRCCATGCANCACCT-3′) to amplify regions V5-V6 of the 16S rRNA sequence with KAPA HiFidelity Hot Start Polymerase. Samples were analyzed with initial cycling conditions of 95 °C for 5 min, with 25 cycles of 98 °C for 20 s, 55 °C for 15 s, and 72 °C for 1 min; followed by the addition of Illumina adapter, barcode sequences and dual index PCR for 10 cycles [33 (link)]. Sequences were purified by gel, pooled, and pair-end sequenced (300 nt read length) using the Illumina MiSeq platform (Illumnia, Inc., San Diego, CA, USA) [33 (link)].