Cecal Content Sample Processing for GC-MS

Sample processing protocols for cecal contents were according to the method described with some minor modifications.^{38 (link)} Briefly, ~30 mg of fresh cecal content was mixed with 300 μL of 1 M NaOH solution, homogenized with 1 mm zirconium beads and centrifuged at 16,000 × g at 4°C for 20 min. A volume of 200 μL of supernatant was collected into an autosampler vial. Then, the residue was re-extracted with 200 μL of cold methanol with the same steps of homogenization and centrifugation. The first supernatant in the sample vial was combined with 200 μL of supernatant from the second extraction. Subsequently, 300 μL of chloroform containing 30 µM indole-d6 was added to the mixed supernatant and samples were shaken for 10 s. Another 400 μL of 50 mM sodium bicarbonate solution was added again, and samples were shaken for another 10 s. A volume of 100 μL chloroform phase was transferred to GC vials for further analysis after centrifugation at 2000 × g for 10 min at 4°C. Indole and 3-methylindole were quantified with an Agilent 7890A gas chromatograph coupled with an Agilent 5975 mass spectrometer (Agilent Technologies Santa Clara, CA, USA).