DNA was extracted from an equal volume of normalized homogenates of cotton rat stool (described in Methods: Enumeration of Lactobacillus) using the DNeasy PowerSoil Kit (Qiagen). qPCR reactions were prepared in duplicate using BioRad iQ Supermix with Invitrogen Sybr Green following the manufacture’s protocol. Universal eubacteria 16S rRNA primers (UniF340, UniR514) [90] equal volumes of extracted DNA, and targeted standards were used to determine copy number per gram of feces. Each qPCR plate included a corresponding extraction negative and a no-template negative control. A serial dilution of standards containing known bacterial copy numbers specific to the primer pair were used as a standard curve as previously described. PCR reactions were run with a 15 s 95 °C melting and 1 min 54 °C annealing step for 40 cycles. Cycle threshold (CT) values were plotted against the standard curve to determine copy number, and figures and statistical testing (unpaired T test) were generated using Prism version 8.

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