16S rRNA Gene Sequencing of Fecal Microbiome

Total genomic DNA was extracted from fecal pellets from a subset of the mice in cohort 1 (chow = 13 mice from four separate cages over two experiments, WD = 13 mice from four separate cages over two experiments, LF/LF = 5 mice from two cages over two experiments) using the DNeasy PowerSoil Kit (Qiagen, Germantown, MD). Modifications to the standard protocol included a 10-min incubation at 65 °C immediately following the addition of the lysis buffer and the use of a bead mill homogenizer at 4.5 m/s for 1 min. The V4 variable region of the 16S rDNA gene was targeted for sequencing (515F: GTGCCAGCMGCCGCGGTAA, 806R: GGACTACHVGGGTWTCTAAT). The target DNA was amplified using 5Prime HotMaster Mix (Quantabio, Beverly, MA). Construction of primers and amplification procedures follow the Earth Microbiome Project guidelines (www.earthmicrobiome.org)^{66 (link)}. Amplified DNA was quantified in a PicoGreen (ThermoFisher Scientific) assay and equal quantities of DNA from each sample was pooled. The pooled DNA was sequenced using a V2 2 × 250 kit on the Illumina MiSeq platform (San Diego, CA) at the University of Colorado Anschutz Medical Campus Genomics and Microarray Core facility.