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assays/MTBLS338_GCMS_Root_met/protocols/Chromatography.md

+<p><strong>LC Chromatography:</strong></p><p>Samples (2.6 μl, full loop injection) were separated at 40 °C on an Acquity UPLC platform (Waters, www.waters.com) equipped with a HSS T3 column (100 × 1.0 mm, particle size 1.8 μm, Waters) applying the following gradient at a flow rate of 150 μl/min: 0–1 min, isocratic 95% A (water/formic acid, 99.9/0.1 (v/v)), 5% B (acetonitrile/formic acid, 99.9/0.1 (v/v)); 1–16 min, linear from 5 to 95% B; 16–18 min, isocratic 95% B; 18–20 min, isocratic 5% B.</p><p><br></p><p><strong>GC Chromatography:</strong></p><p>For GC/MS analysis, 40 µl of the root extract were vacuum-evaporated and subjected to a derivatization with firstly methoxyamine hydrochloride and secondly N,O-bis(trimethylsilyl)trifluoroacetamide. Derivatized samples were injected in a splitless manner into a split/splitless inlet of an Agilent 6890N GC and a ZB-5 column (30 m × 0.25 mm, 0.25 m 95% dimethyl /5% diphenyl polysiloxane film, 10 m integrated guard column, Phenomenex) at 230 °C. The following temperature programme applied: 70 °C/5 min, increasing by 9 °C per min up to 350 °C, 350 °C/5 min, decreasing by 10 °C per min to 330 °C, then 330 °C/5 min.</p>
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assays/MTBLS338_GCMS_Root_met/protocols/Data_transformation.md

+<p><strong>LC/MS:</strong></p><p>Targeted; DataAnalysis 4.0 (Bruker Daltonics) was used for generation of extracted ion chromatograms, deconvolution of compound mass spectra and calculation of elemental compositions. For the relative quantification of compounds extracted, ion chromatograms of quantifier ions were integrated applying QuantAnalysis 2.0 (Bruker Daltonics).</p><p>Untargeted; Raw data files were converted to mzData using CompassXPort (Bruker Daltonics 4.0). Subsequently, the R package XCMS [1] was used for feature detection, alignment and filling of missing values. On this account, features were detected with the help of the centWave algorithm according to Tautenhahn et al. (2008) [2] (sntresh: 5, prefilter: (3,100), ppm: 20, peak width: (5,12), scanrange=c(1,3060) and aligned using the XCMS function group.density (minfrac: 0.75, bw: 2, mzwid: 0.05). Missing values were replaced with the help of the XCMS function fillPeaks.</p><p><br></p><p><strong>GC/MS:</strong></p><p>Vendor file format conversion and baseline correction was performed by MetAlign. Peaks with intensities above 500 were subsequently processed with TagFinder and mass spectral features were grouped according to their retention time. Peak clusters with 3 peaks were extracted.</p><p><br></p><p><strong>Ref:</strong></p><p><strong>[1]</strong> Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G. Anal Chem. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. 2006 Feb; 78(3):779-87. [2] Tautenhahn R, Böttcher C, Neumann S. Highly sensitive feature detection for high resolution LC/MS. BMC Bioinformatics. 2008 Nov; 9:504.</p>
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assays/MTBLS338_GCMS_Root_met/protocols/Extraction.md

+After addition of 200 µl methanol/water, 80/20 (v/v) pre-cooled at –40 °C the samples were immediately vortexed for 15 s, sonicated for 15 min at 20 °C and centrifuged for 10 min at 19000 x g. The supernatants were transferred to new 2 ml tubes and the remaining pellets subjected to a second extraction using 200 µl methanol/water, 80/20 (v/v). The combined extracts were evaporated to dryness in a vacuum centrifuge at 30 °C and reconsituted corresponding to a final concentration of 40 mg root material in 200 µl methanol/water 30/70 (v/v) containing 5 µM 2,4-dichlorophenoxyacetic acid as an internal standard.
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assays/MTBLS338_GCMS_Root_met/protocols/Mass_spectrometry.md

+<p>Eluting compounds were detected in positive and negative ionization mode from m/z 80 – 1000 using a MicroTOF–Q I hybrid quadrupole time-of-flight mass spectrometer equipped with an Apollo II electrospray ion source (Bruker Daltonics, Billerica, MA, USA). Instrument parameters were defined as follows for the positive mode: nebulizer gas, nitrogen, 1.6 bar; dry gas, nitrogen, 6 L/min, 190°C; capillary, –5000 V; end plate offset, -500 V; funnel 1 RF, 200 Vpp; funnel 2 RF, 200 Vpp; in-source CID energy, 0 V; hexapole RF, 100 Vpp; quadrupole ion energy, 5 eV, low mass 55; collision gas, argon; collision energy, 10 eV; collision RF 150 Vpp; transfer time, 70 μs; pre pulse storage, 5 μs; spectra rate, 3 Hz. For the negative ionization mode all parameters were maintained except for the capillary voltage (4000 V).</p><p>All mass spectra were acquired in centroid mode and recalibrated on the basis of lithium formiate cluster ions. EI-MS:</p><p>An Agilent 5975C inert XL MSD Mass Selective Detector was used to detect eluting compounds from m/z 70-600.</p>
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assays/MTBLS338_GCMS_Root_met/protocols/Metabolite_identification.md

+<p><strong>LC/MS:</strong></p><p>The target compounds were obtained from prior experiments, including comparison to authentic standards (MSI level 1) and additional putative identifications (MSI levels 2 and 3).</p><p><br></p><p><strong>GC/MS:</strong></p><p>Metabolites were identified according to matching mass spectra using the Golm Metabolome Database (http://gmd.mpimp-golm.mpg.de/) and the NIST2012.</p>
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assays/MTBLS338_GCMS_Root_met/protocols/Sample_collection.md

+<p>Seeds of the accession Bur-0, Col-0, Can-0, Ct-1, Edi-0, Hi-0, Kn-0, Ler-0, Mt-0, No-0, Oy-0, Po-0, Rsch-4, Sf-2, Tsu-0, Wil-2, Ws-0, Wu-0, and Zu-0 of Arabidopsis thaliana were obtained from the European Arabidopsis Stock Centre and surface sterilized prior to plant cultivation. Then, all accessions were cultivated in a hydroponic system with three independent biological experiments.</p><p><br></p><p>For hydroponic cultivation, a pre- and main culture set-up was used. The pre-culture system consisted of PCR tubes filled with approx. 170 µl agar solution (8% (w/v) GELRITE, 1% (w/v) sucrose) placed in a yellow pipette tip box. After solidification, bottoms were cut and individual seeds were sown on a total of 48 tubes, submerged partially in nutrient solution (half-strength Murashige and Skoog (MS) medium supplemented with 1% (w/v) sucrose and Gamborg’s B-5 vitamins (Duchefa), pH 5.8). Boxes were closed with their lids and sealed with leucoplast (Duchefa). Plants were grown at 22 °C under short-day conditions (8 h light, 130 µmol/m2/s) to prevent early inflorescences until all roots had penetrated the agar plug. After three weeks, the main-culture system was set up. Tubes were transferred into perforated screw caps of 50ml amber bottles (Duran, Wertheim/Germany) filled with approximately 70 ml nutrient solution. Modified MS medium (1.0 mM KH2PO4, 1 mM MgSO4, 0.25 mM K2SO4, 0.25 mM CaCl2, 2 mM NH4-NO3, 0.1 mM Na-Fe-EDTA, 50 µM KCl, 30 µM H3BO3, 5 µM MnSO4,1 µM ZnSO4, 1 µM CuSO4, 0.7 µM NaMoO4, pH 5.8) was used as a culturing medium to enable plant growth over a prolonged period without osmotic stress and sufficient nutrient supply. Medium was exchanged weekly accompanied by a check for microbial contamination after harvest. Glass bottles were kept in sealed plastic boxes (Araponics, Liège/Belgium) with 12 plants each.</p><p><br></p><p>This experimental setup resulted in 228 root samples from individual plants. Single-plant roots were harvested at week 6 and homogenized in liquid nitrogen using a pestle and mortar, resulting in 222 and 210 usable root samples for LC/MS and GC/MS analysis, respectively. Primary root length was determined with a ruler, root fresh weight with fine scales, and results are listed in Table phenotypicCharacteristics.csv.</p>
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