. Briefly, cecal and serum metabolites were extracted by vigorous shaking with methanol containing methionine sulfone and D-camphol-10-sulfonic acid as the internal standards. Then the mixture was cleaned with chloroform and water extraction. The suspension was centrifuged at 4600 ×for 15 min at 4 °C, and the resulting supernatant was transferred to a 5-kDa-cutoff filter column .
. All CE-TOFMS experiments were performed using an Agilent capillary electrophoresis system and Agilent G3250AA LC/MSD TOF system .60 . Annotation tables were produced from standard compound measurements and aligned with the datasets according to similar m/z values and normalized migration time. Then, peak areas were normalized against the internal standards methionine sulfone and CSA for cationic and anionic metabolites, respectively. Amounts of each metabolite were calculated based on their relative peak areas and the concentrations of the standard compounds.
. Concentrations of individual BAs were determined from the peak area in the chromatogram detected with MRM relative to the internal standard, CSA.Organic acid concentrations of cecal contents were determined by gas chromatography–mass spectrometry and homogenized with extraction solution containing 100 μl of internal standard , 50 μl HCl and 200 μl ether. After vigorous shaking using Shakemaster neo at 1500 r.p.m.
. RNA was digested in the sample by RNase A treatment; the resulting DNA sample then was purified again, by another round of phenol/chloroform/isoamyl alcohol treatment.16S rRNA genes in the fecal microbiota DNA samples were analyzed using a Miseq sequencer . The V1–V2 region of the 16 S rRNA genes was amplified from the DNA using a universal bacterial primer set consisting of primers 27Fmod with an overhang adapter and 338R with an overhang adapter .