Neonatal dairy calf gut microbiota-derived ursodeoxycholic acid improves intestinal homeostasis and colitis to attenuate diarrheagenic Escherichia coli infection
Metabolomics preprocessed data, the data set is related to figure 2, Extended Data figure S3.
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Feces were thawed on ice-bath to diminish degradation. About 5 mg of each lyophilized sample was weighted and transferred to a new 1.5 mL tube. Then 25 μL of water was added and the sample was homogenated with zirconium oxide beads for 3 minutes and 120μL of methanol containing internal standard was added to extract the metabolites. The sample was homogenated for another 3 minutes and then centrifuged at 1,800 g for 20 minutes. The 20 μL of supernatant was transferred to a 96-well plate and following procedures were performed on a Eppendorf epMotion Workstation (Eppendorf Inc., Humburg, Germany). 20 μL of freshly prepared derivative reagents was added to each well. The plate was sealed and the derivatization was carried out at 30°C for 60 min. After derivatization, sample was further diluted by adding 330 μL of ice-cold 50% methanol solution. Then, the plate was stored at -20°C for 20 minutes and followed by 4,000 g centrifugation at 4℃ for 30 minutes. 135 μL of supernatant was transferred to a new 96-well plate with 10 μL internal standards in each well. Serial dilutions of derivatized stock standards were added to the left wells. Finally, the plate was sealed for LC-MS analysis. Metabolite profiling and data processing were performed using a ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA). The analytes were separated on an ACQUITY UPLC BEH C18 1.7 µM VanGuard pre-column (2.1×5 mm) and ACQUITY UPLC BEH C18 1.7 µM analytical column (2.1×100 mm). Mobile phases were used as carried liquid at a constant flow rate of 0.4 mL/min. The source and desolvation temperatures of was set at 150℃ and 500℃, respectively. Each sample was analysed by UPLC-MS/MS in both negative and positive ionization modes to acquire metabolite profiles. The raw data files generated by UPLC-MS/MS were processed using the MassLynx software (version 4.1, Waters, Milford, MA, USA) to perform peak integration, calibration, and quantitation for each metabolite. The analysis order of all test samples was randomized. Quality control (QC) samples were obtained by mixing a small aliquot of each biological sample in the study set. The pooled QC represented both the sample matrix and metabolite composition of the samples. The raw pooled QC mixture were used to produce multiple QC samples that would be analyzed during the whole injection sequence. In metabolomics the application of QC samples provides a mechanism to evaluate the quality and assess the analytical variance of the aquired data. The self-developed platform iMAP (version 1.0, Metabo-Profile, Shanghai, China) was used for statistical analyses, including PCA, OPLS-DA, univariate analysis and pathway analysis.