Microbial Community Analysis of Gut Samples

Genomic DNA samples were extracted using the CTAB method⁵⁷ on five individual gut homogenates from each biological replicate and treatment. The quantity and quality of extracted gDNA were determined using a NanoDrop Spectrophotometer (Eppendorf AG, Germany) and stored at −20 °C prior to the analysis. The pyrosequencing of V2-V3 hyper-variable regions of the 16S rRNA gene amplicons was carried out on a 454 GS FLX Titanium system (Roche, Penzberg, Germany)^{60 (link),62} . Universal barcoded primers Ba27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and Ba519R (5′-TAT TAC CGC GGC KGC TG-3′) containing A and B sequencing adaptors (454 Life Sciences), key sequences and multiplex identifiers were used to prepare the pyrosequencing amplicons^{63 (link)}. Before pyrosequencing, amplicons were purified from gel using AxyPrep DNA gel extraction kit (Axygen Scientific, Inc.), quantified using Quant-iT Pico Green dsDNA assay (Invitrogen, Germany) and pooled in an equimolar ratio before being subjected to emulsion PCR and breaking. GS FLX Titanium chemistry was used to perform pyrosequencing from A-end according to the supplier protocol.