The same material used for transcriptome sequencing was used for metabolome sequencing, with six replicates per treatment. Shanghai Lumine Biotechnology Co. carried out the metabolite extraction and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. The metabolic profiles in ESI-positive and ESI-negative ion modes were analyzed using an AB SCIEX Triple TOF 5600 system (AB SCIEX, Framingham, MA, USA) and an ACQUITY UHPLC system (Waters Corporation, Milford, MA, USA). For both positive and negative ion modes (1.7 µm and 2.1 × 100 mm), an ACQUITY UPLC BEH C18 column was utilized. Progenesis QI data processing software (Waters Corporation, Milford, MA, USA) was used to process the metabolite data, and the identified metabolites were analyzed using custom databases for metabolite identification along with public databases (e.g., http://www.lipidmaps.org/ , accessed on 1 April 2019) and http://www.hmdb.ca/ (accessed on 1 April 2019) that included public databases for metabolite identification. The criteria used to establish differential metabolites were VIP value > 1 and p-value < 0.05 [33 (link)].
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