Prepare samples for metabolomics
For RMBL and Kessler experiments, plant leaf and root samples (100 mg) were weighed into 1.5 mL centrifuge tubes. Added 1.3 mL extraction solvent (40:40:20 HPLC grade methanol, acetonitrile, water with 0.1% formic acid) pre-chilled to 4°C. Samples vortexed, extracted for 20 min at 4°C while shaken, centrifuged for 5 min (16.1 rcf) at 4°C. Supernatant transferred and extraction repeated twice more. Final supernatant centrifuged and 1.2 mL transferred to vials, dried under N₂ stream, resuspended in 300 μL sterile water.
Quantities: 100 mg plant tissue, 1.3 mL extraction solvent, final 300 μL resuspension volumeDuration: 20 min extraction at 4°C, repeated 3 times total, 5 min centrifugation stepsConditions: 4°C extraction temperature, orbital shaking
Equipment: centrifuge tubes, HPLC grade solvents, vortex, orbital shaker, centrifuge, N₂ stream
Mass spectrometry analysis
Samples analyzed with 10 μL injection through Synergi 2.5 μm reverse-phase Hydro-RP 100, 100 x 2.00 mm LC column at 25°C. Eluted into Exactive™ Plus Orbitrap Mass Spectrometer through 0.1 mm internal diameter fused silica capillary tube. Full scan mode with negative ionization, window 85-1000 m/z, spray voltage 3 kV, nitrogen sheath gas 10 psi flow rate, capillary temperature 320°C, AGC target 3e6, resolution 140,000, scan window 85-800 m/z (0-9 min) and 110-1000 m/z (9-25 min).
Quantities: 10 μL injection volume, 85-1000 m/z mass range, 3 kV spray voltage, 10 psi gas flow, 320°C capillary temperatureDuration: 25 minute gradient run per sampleConditions: 25°C column temperature, negative ionization mode