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Pipeline publication

[…] soflurane, perfused with cold saline and then decapitated. Subsequently, brains were dissected and cortices were snap frozen in liquid nitrogen. After this step, sample preparation, data acquisition, and data processing was performed by the West Coast Metabolomics Center (UC-Davis) as previously described (Fiehn et al., ). Briefly, this non-targeted primary metabolism platform was achieved by gas chromatography—time of flight—mass spectrometry (GC-TOF-MS). Mass spectrometer (Leco Pegasus IV, St. Joseph, MI) settings: unit-mass resolution at 17 spectra s−1 from 80 to 500 Da at −70 eV ionization energy and 1800 V detector voltage with 230°C transfer line and 250°C ion source. Data processing: ChromaTOF vs. 2.32 without smoothing, a 3 s peak width, baseline subtraction above noise level, automatic mass spectral deconvolution and peak detection at 5:1 (signal/noise)., Peak intensity values normalized to tissue weight were used for statistical analysis in MetaboAnalyst 3.0 (Xia and Wishart, ). For normalization, values were log transformed and auto scaled. No filtering was applied given the relatively small number of features identified. Compound names were used for input into pathway and enrichment analysis tools. For Pathway Analysis, which integrates enrichment and pathway topology, over representation was achieved by Fisher's Exact Test and pathway topology by out-degree centrality. Two analyses were run in Enrichment Analysis, which performs metabolite set enrichment analysis (MSEA). Metabolites were compared with (1) pathway-associated metabolite sets, based on normal metabolic pathways and (2) predicted metabolite sets, containing metabolites predicted to be changed in the case of dysfunctional enzymes using genome-scale network model of human metabolism. Integrated Pathway Analysis was carried out using hypergeometric test for enrichment, degree centrality for topology and the gene-metabolite database option. Measured metabolite names were used as input for MetaMapR (Grapov et al., ), to build a network that displays structural similarity of metabolites based on their PubChem substructure fingerprints. The network was then exported and visualized using Cytoscape (Kohl et al., ). All structurally annotated metabolites measured were used to generate structural connections in MetaMapR; however, not all metabolites had structural connections and thus not all metabolites are represented in the network. The full list of metabolites and their metrics can be found in Table ., Real-time qPCR was conducted as described previously (Morris-Blanco et al., ). In short, TRIzol (Life Technologies) homogenized hippocampi, from the same mice as metabolomics samp […]

Pipeline specifications

Software tools ChromaTOF, MetaboAnalyst, MetaMapR