Feces collection and DNA extraction

Twenty eight mice (6 months-old) were divided in: WT males (WTM), WT females (WTF), KO males (KOM), and KO females (KOF). All of them were treated with doxycycline, were fed the same diet, housed in the same room under specific-pathogen-free (SPF) conditions, and maintained by the same personnel in order to normalize their microbiota. Fresh fecal contents were collected from each animal and weighed. DNA was subsequently extracted from fecal microbiota using the DNeasy Blood & Tissue Kit (Qiagen, Venlo, Netherlands). DNA purity and concentration were determined by a Nanodrop spectrophotometer (ND-1000; Thermo Fisher Scientific, Waltham, MA).