4. Liquid nitrogen quenching - 70% ethanol extraction
The quenching step was performed as described above. Then the sample was added with 3 mL of 70% ethanol (75℃), and the cells were scraped gently and fully with a cell scraper. The cell suspension was harvested into a 10 mL centrifuge tube and 100 μL internal standards were added. The cell suspension was quickly put in 75°C water for 3 min, then centrifuged at 1000×g for 10 min and the supernatant was removed. The cell pellet was resuspended in a 3 mL of the same ethanol solution and the extraction repeated. Finally, the cell pellet was resuspended in 1 mL ice-cold milliQ water followed by 75°C water bath for 3 min. The ethanol and water extracts were pooled together, centrifuged at 12000×g for 10 min and the supernatant was collected and stored at -80°C.