Metabolomics Data Supplementary To: The pioneering gut microbiome acquired via different delivery modes in neonates shape distinct immune and metabolic environments

Fecal Liquid Chromatography Mass Spectrometry (LC-MS/MS) Based MetabolomicsTo each frozen fecal pellet, 400 ul methanol was added followed by 400 ul water and 400 ul chloroform. Samples were agitated for 30 mins at 4°C then centrifuged at 16,000<i>g </i>for 20 mins. 200 ul of the aqueous layer was collected and diluted 5X in 50% methanol and prepared for LC/MS injection. Samples were separated using a Sciex ExionLC AC system and measured using a Sciex 5000 QTRAP or Sciex 65000+ QTRAP mass spectrometer. Polar metabolites were analyzed using ion paired method as previously published with the following modifications⁶⁴. Quality control samples to control for signal stability were injected every 10 injections. Waters Atlantis T3 column was used to separate samples with a binary gradient from 5 mM tributylamine, 5 mM acetic acid in 2% isopropanol, 5% methanol, 93% water (v/v) to 100% isopropanol over 15 minutes. Two distinct multiple-reaction monitoring (MRM) pairs in negative mode were used for each metabolite when possible and relative quantification was performed.