Antibiotic-induced gut microbiota depletion study.

We used a gut microbiota depletion procedure described by Staley et al⁵⁹. Female LDLr−/− mice (n = 35), 8–20 weeks of age were randomized twice between 7 cages in groups of 5 mice/cage to equalize the microbiota and even out age spread. Housing units (cage, bedding, and water bottles) were autoclaved over the entire course of the study. Food was irradiated by vendors. Mice were fed standard chow diet (PicoLab Rodent Diet 20, #5053, % Kcal from: protein 24.65%, carbohydrate 62.14 %, fat 13.20 %) prior to the study and then switched to a high fat diet (Envigo #TD.94059, % Kcal from: protein 20.4 %, carbohydrate 42.7 %, fat 36.9 %) on day −21. The antibiotic cocktail was prepared fresh daily one day prior to consumption, stored at 4°C, and replaced daily. The cocktail consisted of three nonabsorbable antibiotics each at 1 mg ml⁻¹ in the drinking water (ertapenem sodium salt, neomycin sulfate, vancomycin hydrochloride). Peptides were dissolved in sterile 1.2% sucrose prior to the study and stored at −20°C and then diluted with 10X PBS to final 1X. PBS was used as vehicle control. Final concentrations of peptide were 180 μM in drinking water (corresponding to a dose of ~30 mg kg⁻¹ day⁻¹). The timeline of the study is shown in Supplementary Figure 2. Blood and fecal samples were collected at each timepoint and mice were weighed. One cage received no treatment as a control. DNA was isolated from the fecal samples using a DNeasy Powersoil kit (Qiagen). Genomic DNA was measured using a nanodrop 2000. 5 ng of gDNA was amplified using forward and reverse 16Sv3–4 primers (as described elsewhere). Amplicon concentrations were measured using a Qubit dsDNA HS (High Sensitivity) Assay Kit (Thermo Fisher). Equal amounts of each amplicon were combined into one of two libraries (each library containing either 2 or 3 of the 5 replicates from each treatment group, n = 112 or 114 samples, respectively) and cleaned up using Ampure XP beads (Beckman Coulter). Purity was checked using Bioanalyzer High Sensitivity DNA kit (Agilent) and the libraries were sequenced using an Illumina MiSeq (2×300 kit) using a full flow cell run with 20% added PhiX.