Illumina MiSeq paired end sequencing; 16SrRNA analysis from wildtype mice that were either fed or not with low protein diets for 4 weeks
16SrRNA analysis from wildtype mice that were either fed or not with low protein diets for 4 weeks
The mice were sacrificed and the fecal samples were collected. Mice were housed in an enhanced barrier specific pathogen-free facility under a strict 12-hour light cycle. Mice were maintained on a C57BL/6 background. All study protocols were approved by the Washington University Animal Studies Committee. Mice were fed irradiated standard diet (PicoLab Rodent Chow 20, Purina Mills; containing 20% protein), isocaloric protein-free diet (Envigo # TD.99328), or isocaloric 6% protein diet (Envigo # TD.90016). DNA was isolated using Qiagen QIAmp DNA Stool Kit, according to the kit protocol. Total DNA concentration and purity was obtained from an absorbance ratio at 260nm and 280nm. 14 PCR amplicons, representing all 9 16S variable regions, were constructed using the Fluidigm Access Array System. 5ng/ul of DNA were input into each reaction. The sample inlets consisted of 1X High Fidelity FastStart Reaction Buffer without MgCl2 (Roche), 4.5nM MgCl2 (Roche), 5% DMSO (Roche), 200uM PCR Grade Nucleotide Mix (Roche), 0.05 U/L 5 U/L FastStart High Fidelity Enzyme Blend (Roche), 1X Access Array Loading Reagent (Fluidigm), 1ul DNA, and water. The primers were added to the assay inlets at 200nM forward and reverse primers with 1X Access Array Loading Reagent. The samples were harvested after PCR amplification was performed on the BioMark HD system from Fluidigm. Each sample was harvested and indexed using unique 10 base pair sequences with 14 rounds of PCR to incorporate each index sequence. All samples were pooled into 48 sample libraries and cleaned using bead purification.