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MB Sample ID: SA026388

Local Sample ID:50.1
Subject ID:SU000530
Subject Type:Date palm fruit
Subject Species:Phoenix dactylifera
Taxonomy ID:42345
Species Group:Plant

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Sample Preparation:

Sampleprep ID:SP000537
Sampleprep Summary:Sample pre-processing for DS1: 50 mg of the peel and flesh of the date fruits were flash frozen in liquid nitrogen and extracted as previously described (PMID:21699588). Sample pre-processing for DS2 The beads were added to the pre-weighed frozen samples together with water (8 µL of per mg of sample) for homogenisation. The samples were continuously stirred on the GenoGrinder (Glen Mills GenoGrinder 2000, Germany) at 1000 strokes per minute for five minutes, to ensure complete homogenization. 100 µL of aliquot from each sample was transferred to the plates. The samples were loaded on three plates. To each sample, 450 µL of extraction solvent (MeOH with 10 µL /ml chlorophenylalanine, 2.5 µL /ml 2-fluorophenylglycine, 25 µg/ml d-6 cholestrol and 25 µL /ml tridecanoic acid) was added. The samples were then shaken on the GenoGrinder (GenoGrinder, Spex, USA) at 675 strokes per min for two minutes and centrifuged at 2000 rpm for 5 minutes on a Beckman centrifuge (Beckman GS-6R Centrifuge, USA) at 40C. The extracted samples were divided into equal parts for metabolomics analysis on the Gas Chromatography Mass Spectrometry (GC/MS) and the Orbitrap Elite accurate Liquid Chromatography Mass Spectrometry2 (LC-MS-MS) platforms. Four sets of samples were prepared by the Hamilton robot (Hamilton Star, Germany) by transferring 110 µL aliquots from each well to three PCR plates, each for LC positive, LC negative, replicate set and one to 250 µL auto sampler vial inserts for GC. All samples were dried for 120 minutes by using a Zymark Turbovap 96 (Zymark Turbovap, USA) followed by overnight incubation in a drybox to ensure optimal dryness of the sample. Sample processing DS1 and DS2 Metabolite measurements with Ultrahigh Performance Liquid Chromatography/Mass Spectroscopy (UPLC/MS/MS): The UPLC/MS/MS analysis was based on the Waters ACUITY ultra performance liquid chromatography (Waters Corporation, USA) and the ThermoFischer Scientific Orbitrap Elite high-resolution accurate mass spectrometer (Thermo Fischer Scientific Inc., USA) equipped with a heated electrospray ionization (HESI) source and an Orbitrap mass analyzer. The dried sample extracts for the LC positive and LC negative mode were reconstituted in acidic and basic LC- compatible solvents. Two independent injections were performed on each sample using separate dedicated columns for optimized acidic positive ions and the other for optimized basic negative ions. The acidic samples were reconstituted by gradient elution of water and methanol containing 0.1 % formic acid whereas; the basic samples were reconstituted by gradient elution of water and methanol containing 6.5mM ammonium bicarbonate (PMID: 19624122). The mass spectra analysis alternated between MS and data dependent MS2 scans using dynamic exclusion. Metabolite measurements with GC/MS: The samples assigned for the GC/MS analysis were further dried under vacuum desiccation for an entire day and derivatized under dried nitrogen using bistrimethyl-silyl-trifluoroacetamide (BSTFA). The GS/MS analysis was based on a Thermo FinniganTM TRACETM DSQTM (ThermoFinnigan, USA) fast-scanning single –quadrupole mass spectrophotometer using electron impact ionization source. The GC column was 5% phenyl and the temperature ramp range was from 40 to 3000 C in a time span of 16 minutes.