Short-chain Acyl-CoA Extraction

Extractions were performed as described previously³⁴. Briefly, lymphocytes were centrifuged at 1200 rcf for 5 min. Cell pellets were resuspended in 750 µl of ice-cold 10% trichloroacetic acid and pulse-sonicated using a sonic dismembrator (Fisher). The samples were centrifuged at 15,000 rcf for 15 min and the supernatants were purified by solid phase extraction. Briefly, Oasis HLB 1-ml (30 mg) solid-phase extraction columns were conditioned with 1 ml methanol, followed by 1 ml of H₂O. The supernatants were applied to the column and washed with 1 ml of H₂O. The analytes were eluted in methanol containing 25 mM ammonium acetate. The eluates were dried under N₂ gas and resuspended in 50 µl of 5% 5-sulfosalicylic acid. Samples were analyzed by LC/ESI/MS/MS using an Ultimate 3000 Quaternary UHPLC coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) operating in the positive ion mode as previously described³⁵.