Computational protocol: Arachidonic Acid Metabolism Pathway Is Not Only Dominant in Metabolic Modulation but Associated With Phenotypic Variation After Acute Hypoxia Exposure

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Protocol publication

[…] Metabolic profiling was performed, and metabolic data were obtained as previously described (Liao et al., ). Briefly, blood was drawn from a peripheral vein in the morning soon after waking before ascending to high altitudes and after 3 days high-altitude exposures. Blood samples were centrifuged to separate serum at 14,000 × g for 15 min at 4°C. Then, these plasma samples were delivered to Chongqing for further metabolomics analysis in the courier filed with dry ice.Metabolomics analysis was conducted on an Agilent 1290 Infinity LC system (Agilent, Santa Clara, CA, USA). Chromatographic separations were performed on an ACQUITY UHPLC HSS T3 C18 column (2.1 × 100 mm, 1.8 μm; Waters, Milford, Ireland) at 45°C. The metabolomics detection was performed totally in accordance to the manufacturers' protocols of all devices. Raw LC-MS data were converted to mzData formats via Agilent MassHunter Qualitative software (Agilent, Santa Clara, CA, USA). The program XCMS (version 1.40.0) (https://xcmsonline.scripps.edu/) was used to preprocess the raw data, including peak detection, peak matching, matched filtration, and nonlinear alignment of data, with the default parameters (and snthresh, 5; bw, 10; fwhm, 10).The resulting matrix from 53 subjects comprising retention time, mass-to-charge ratio (m/z), and normalized ion intensities was introduced into the subsequent analysis based on R platform (https://www.r-project.org). The model of partial squares discriminant analysis (OPLS-DA) was carried out to separate pre- and post-hypoxia in metabolic profiling. Variable importance project (VIP) was used to reveal discriminatory metabolites which were enrolled in the classification effects of hypoxia was calculated. In addition, adjusted p-value from the paired t-test and fold-change value (FC) were executed to discovery dominant metabolites. Pathway analysis was established by MetaboAnalyst platform (http://www.metaboanalyst.ca). [...] To improve the reliability of the integrated analysis to uncover interactions at metabolic and transcriptional level, 11 individuals were randomly selected and subjected to metabolomic and RNA-seq detection (Li et al., ). RNA sequencing was performed as previously described (Liu et al., ) with minor modifications. Briefly, after extraction, total RNA was quantified by spectrophotometer (NanoDrop 200, Thermo Scientific, Delaware, USA). mRNA-seq libraries were constructed according to the TruSeq RNA Sample Prep Kit v2 (Illumina) and sequenced on an Illumina HiSeq 2000 sequencer, following the manufacturer's instructions. after filtered, the clean reads were aligned to human genome hg19 based on HISAT and assembled by Stringtie (Li et al., ). The expression value of each transcript was subsequently calculated based on methods of Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). In addition, GSE52209 datasets of blood samples from individuals suffering from adaptation or mal-adaptation to high altitude exposure were downloaded from Gene Expression Omnibus (GEO) datasets. After annotation, limma package was employed to normalize and detect the significantly differential genes involved in diverse response manner to hypoxia (Ritchie et al., ). ClusterProfiler package was used to determine enriched Gene Ontology (GO) terms in differentially expressed gene sets (Yu et al., ). […]

Pipeline specifications

Software tools HISAT, StringTie, limma, clusterProfiler
Applications Genome annotation, RNA-seq analysis
Organisms Hemisus marmoratus
Diseases Altitude Sickness
Chemicals Oxygen, Arachidonic Acid