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MB Sample ID: SA189027

Local Sample ID:	71_071
Subject ID:	SU002098
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702

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Subject:

Subject ID:	SU002098
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
71_071	SA189027	FL023225	leaves	organ
71_071	SA189027	FL023225	Col-0_L	genotype

Collection:

Collection ID:	CO002091
Collection Summary:	Samples were collected from live plants and frozen in liquid nitrogen
Sample Type:	Plant

Treatment:

Treatment ID:	TR002110
Treatment Summary:	four genotypes, each with three tissue types, 6, no treatment, just genotype and tissue type difference.

Sample Preparation:

Sampleprep ID:	SP002104
Sampleprep Summary:	Extraction of Plant Samples: Leaves 1. References: Fiehn O (2006) Metabolite Profiling in Arabidopsis. In: Arabidopsis Protocols 2nd edition. Salinas J, Sanchez-Serrano JJ (eds.), Methods in Molecular Biology ser., Humana Press, Totowa NJ, 439-447 Weckwerth W, Wenzel K, Fiehn O (2004) Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks. Proteomics 4, 78-83 2. Starting material: Arabidopsis rosette leaves: 20±5 mg fresh weight sample OR 2 mg dry weight sample Rice leaves: 10±3 mg fresh weight sample OR 1 mg dry weight sample Other plant organs not validated but advised to use this SOP in a similar manner 3. Equipment: Eppendorf pipettes: 1-200µL and 100-1000µL Eppendorf tubes 2.0mL, uncoloured (Cat. No. 022363204 ) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Precision balance with accuracy ± 0.1 mg ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer, VWR. Speed vacuum concentration system (Labconco Centrivap cold trap) Ball mill MM301 (Retsch corp.) Metal balls (Retsch 3 mm I.D. Cat. No 22.455.0002 or 5 mm I.D. Cat. No 22.455.0003) Large tweezers Dewar cold gloves 2 liquid nitrogen dewars pH paper 5-10 (EMD Chem. Inc.) Crushed ice Nitrogen line with pipette tip Cork borer (Ø diameter of cylinder related to the amount to be used. 4 mm I.D. for Arabidopsis leaves.) 4. Chemicals Methanol, LCMS grade Chloroform, HPLC grade Liquid nitrogen Water, Millipore (pure) 5. Harvest Procedure NOTE: No weighing of plant material needed unless the experimental design suggests marked changes for the ratio of leaf disk diameter to fresh weight, e.g. in drought stress experiments. In such cases, leaves need to be lyophilized and weighed prior to extraction Label Eppendorf tubes according to the sample name defined in SetupX and put a grinding ball in each. Using the cork borer take one or two disks per leaf and immediately transfer to an Eppendorf tube. Close the tube and immediately place into the dewar of liquid nitrogen. 6. Preparation of extraction mix and material Check the pH of methanol (pH7) Switch on the ThermoElectron Neslab RTE 740 cooling bath at to pre-cool at –18°C to -22°C Make the extraction solution by mixing methanol, chloroform, and water in proportions of 5 : 2 : 2 Rinse the extraction solution for 5 min with nitrogen, making sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution. 7. Homogenisation and extraction Take the Eppendorf tubes from the liquid nitrogen and place into the tube-holder of the grinder being careful to compensate for weight, maintaining equilibrium. Shake for 30s at a frequency of 25 s-1 and check that the leaves have been ground into a fine powder. Repeat if necessary, submerging in liquid nitrogen first. After grinding add 1mL of pre-chilled extraction solution to each tube one by one to prevent even partial thawing of the sample. Store all samples on ice while finishing this step. Vortex the sample for 10s and shake on the Orbital Mixing Chilling/Heating Plate for 6 minutes at 4°C Centrifuge for 2min at 14000 rcf using the centrifuge Eppendorf 5415 D. Remove the whole supernatant in two 500uL portions, saving one as a backup. Dry one portion in the Labconco Centrivap cold trap concentrator to complete dryness and submit for derivitization. 8. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 8. Quality assurance For each sequence perform one blank negative control by applying the total procedure (i.e. all materials and plastic ware) without biological sample. Also perform all tasks once samples/standards have come to room temperature. Finally, include a pure MSTFA vial for liner conditioning steps. 9. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Sampleprep ID:

SP002104

Sampleprep Summary:

Extraction of Plant Samples: Leaves 1. References: Fiehn O (2006) Metabolite Profiling in Arabidopsis. In: Arabidopsis Protocols 2nd edition. Salinas J, Sanchez-Serrano JJ (eds.), Methods in Molecular Biology ser., Humana Press, Totowa NJ, 439-447 Weckwerth W, Wenzel K, Fiehn O (2004) Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks. Proteomics 4, 78-83 2. Starting material: Arabidopsis rosette leaves: 20±5 mg fresh weight sample OR 2 mg dry weight sample Rice leaves: 10±3 mg fresh weight sample OR 1 mg dry weight sample Other plant organs not validated but advised to use this SOP in a similar manner 3. Equipment: Eppendorf pipettes: 1-200µL and 100-1000µL Eppendorf tubes 2.0mL, uncoloured (Cat. No. 022363204 ) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Precision balance with accuracy ± 0.1 mg ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer, VWR. Speed vacuum concentration system (Labconco Centrivap cold trap) Ball mill MM301 (Retsch corp.) Metal balls (Retsch 3 mm I.D. Cat. No 22.455.0002 or 5 mm I.D. Cat. No 22.455.0003) Large tweezers Dewar cold gloves 2 liquid nitrogen dewars pH paper 5-10 (EMD Chem. Inc.) Crushed ice Nitrogen line with pipette tip Cork borer (Ø diameter of cylinder related to the amount to be used. 4 mm I.D. for Arabidopsis leaves.) 4. Chemicals Methanol, LCMS grade Chloroform, HPLC grade Liquid nitrogen Water, Millipore (pure) 5. Harvest Procedure NOTE: No weighing of plant material needed unless the experimental design suggests marked changes for the ratio of leaf disk diameter to fresh weight, e.g. in drought stress experiments. In such cases, leaves need to be lyophilized and weighed prior to extraction Label Eppendorf tubes according to the sample name defined in SetupX and put a grinding ball in each. Using the cork borer take one or two disks per leaf and immediately transfer to an Eppendorf tube. Close the tube and immediately place into the dewar of liquid nitrogen. 6. Preparation of extraction mix and material Check the pH of methanol (pH7) Switch on the ThermoElectron Neslab RTE 740 cooling bath at to pre-cool at –18°C to -22°C Make the extraction solution by mixing methanol, chloroform, and water in proportions of 5 : 2 : 2 Rinse the extraction solution for 5 min with nitrogen, making sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution. 7. Homogenisation and extraction Take the Eppendorf tubes from the liquid nitrogen and place into the tube-holder of the grinder being careful to compensate for weight, maintaining equilibrium. Shake for 30s at a frequency of 25 s-1 and check that the leaves have been ground into a fine powder. Repeat if necessary, submerging in liquid nitrogen first. After grinding add 1mL of pre-chilled extraction solution to each tube one by one to prevent even partial thawing of the sample. Store all samples on ice while finishing this step. Vortex the sample for 10s and shake on the Orbital Mixing Chilling/Heating Plate for 6 minutes at 4°C Centrifuge for 2min at 14000 rcf using the centrifuge Eppendorf 5415 D. Remove the whole supernatant in two 500uL portions, saving one as a backup. Dry one portion in the Labconco Centrivap cold trap concentrator to complete dryness and submit for derivitization. 8. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 8. Quality assurance For each sequence perform one blank negative control by applying the total procedure (i.e. all materials and plastic ware) without biological sample. Also perform all tasks once samples/standards have come to room temperature. Finally, include a pure MSTFA vial for liner conditioning steps. 9. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Combined analysis:

Analysis ID	AN003286
Analysis type	MS
Chromatography type	GC
Chromatography system	Gerstel CIS4 –with dual MPS Injector/ Agilent 6890 GC- Pegasus III TOF MS
Column	Rtx-5Sil MS
MS Type	EI
MS instrument type	GC-TOF
MS instrument name	Leco Pegasus IV TOF
Ion Mode	UNSPECIFIED
Units	normalized peak height

Chromatography:

Chromatography ID:	CH002426
Chromatography Summary:	Gas Chromatography conditions: A 30 m long, 0.25 mm i.d. Rtx-5Sil MS column (0.25 μm 95% dimethyl 5% diphenyl polysiloxane film) with additional 10 m integrated guard column is used (Restek, Bellefonte PA). 99.9999% pure Helium with built-in purifier (Airgas, Radnor PA) is set at constant flow of 1 ml/min. The oven temperature is held constant at 50°C for 1 min and then ramped at 20°C/min to 330°C at which it is held constant for 5 min.
Instrument Name:	Gerstel CIS4 –with dual MPS Injector/ Agilent 6890 GC- Pegasus III TOF MS
Column Name:	Rtx-5Sil MS
Chromatography Type:	GC

MS:

MS ID:	MS003057
Analysis ID:	AN003286
Instrument Name:	Leco Pegasus IV TOF
Instrument Type:	GC-TOF
MS Type:	EI
MS Comments:	Mass spectrometer settings: A Leco Pegasus IV time of flight mass spectrometer is controlled by the Leco ChromaTOF software vs. 2.32 (St. Joseph, MI). The transfer line temperature between gas chromatograph and mass spectrometer is set to 280°C. Electron impact ionization at 70V is employed with an ion source temperature of 250°C. Acquisition rate is 17 spectra/second, with a scan mass range of 85-500 Da.
Ion Mode:	UNSPECIFIED