title: Parental dietary fat intake alters offspring microbiome and immunity - pup from breeders fed western diet
design: The samples are surgically/sterilely resected stool from the cecum of 5-7 week old pups. The pups came from breeders that were exposed to different diets. The breeders were placed on the diets one day before placement in breeding cages and remained on the diets throughout the study. All pups from all breeders were weaned onto the low fat control diet. The samples were from pups that thus, were exposed to these diets in utero and during nursing. Therefore, the variable under investigation in the study involving these cecal content samples was the dietary exposure from the breeders/parents during gestation and lactation. The Western Diet (WD) group came from pups who's breeding parents were fed a diet with 40% kcal from fat, much from saturated fat with an over-representation of omega-6. DNA was extracted from sterilely excised cecal stool pellets using QIAamp DNA stool mini kit (Qiagen). For sequencing of 16S rDNA, amplicon libraries were prepared from sample DNA using Accuprime High Fidelity Taq polymerase (Invitrogen) and universal primers flanking variable regions V1 (primer 27F; 5’-AGAGTTTGATCCTGGCTCAG-3’) and V3 (primer 534R; 5’- ATTACCGCGGCTGCTGG-3’). For each sample, the universal primers were tagged with unique sequences ("barcodes") to allow for multiplexing/demultiplexing. PCR products were then purified using the Agencourt Ampure XP Kit (Beckman Counter Genomics) and quantitated using the QuantIT dsDNA High-Sensitivity Assay Kit (Invitrogen). Approximately equivalent amounts of each PCR product were then pooled and purified with a Qiagen minElute column (Qiagen) into 30 microliter TE buffer prior to sequencing at the NIH Intramural Sequencing Center. Sequencing was done on a 454 FLX instrument using Titanium chemistry. Flowgrams were processed using the 454 Basecalling pipeline.