Methanol-based Metabolite Extraction

The extraction protocol used was taken from Bell et al. (2015) (link). Briefly, two experimental replicates of each biological rep (n = 6) were prepared as follows: 40 mg of ground powder was heated in a dry-block at 75°C for 5 min; 1 mL of preheated 70% (v/v) methanol (70°C) was then added to each sample and placed in a water bath for 20 min at 70°C. Samples were centrifuged for 5 min (12,000 rpm, 20°C) to collect loose material into a pellet. The supernatant was then filtered using 0.22 μm Acrodisc syringe filters with Supor membrane (hydrophilic polyethersulfone; VWR, Lutterworth, United Kingdom) into fresh Eppendorf tubes. Samples were frozen at −80°C until analysis by LC–MS.