Masshunter workstation quantitative analysis for qqq version 10

Polar metabolites from samples treated in biological triplicates were extracted using 80% v/v methanol/water and normalized using protein quantification from an additional sample from each condition. Equal estimated amounts of metabolites were dried using a SpeedVac vacuum concentrator, reconstituted in 50% v/v methanol in water, and analyzed by LC-MS as previously described^{92 (link)}. Data were analyzed as previously described^{92 (link)} with the Agilent MassHunter Workstation Quantitative Analysis for QQQ version 10.1 Build 10.1.733.0. However, metabolite abundance levels were not divided by the median levels across the samples. No post-detection normalization was performed to avoid assuming linearity of signal. Raw values of each metabolite measured are provided in Supplementary Table 4. Heatmaps were generated using the Morpheus Matrix Visualization and analysis tool (https://software.broadinstitute.org/morpheus).

The collected supernatants were dried using SpeedVac Vacuum Concentrator, reconstituted with 50% v/v methanol in water, and analysed by LC–MS, as described in detail previously^{47 (link)}. Data were analysed with Agilent Masshunter Workstation Quantitative Analysis for QQQ version 10.1, build 10.1.733.0.