Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA035043

Local Sample ID:	784_B_1
Subject ID:	SU000645
Subject Type:	Photosynthetic organism
Subject Species:	Chlamydomonas reinhardtii
Taxonomy ID:	3055
Genotype Strain:	Wild Type
Species Group:	Microorganism

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Subject:

Subject ID:	SU000645
Subject Type:	Photosynthetic organism
Subject Species:	Chlamydomonas reinhardtii
Taxonomy ID:	3055
Genotype Strain:	Wild Type
Species Group:	Microorganism

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
784_B_1	SA035043	FL007489	WD10784	Class

Collection:

Collection ID:	CO000639
Collection Summary:	Cells were pre-grown to mid-log phase and treated with 5 selected compounds (final concentration 5 µM) with an initial cell density of 1.0 x 106 cells/mL (100 mL volume; in triplicate) and allowed to grow for 72 h. After 72 h of growth, cells were harvested, media removed and freeze-dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min
Sample Type:	Algae

Treatment:

Treatment ID:	TR000659
Treatment Summary:	Cells were treated either with compounds (5 uM) final concentration in DMSO or DMSO alone (in case of control). Mid-log phase cells were used as starter culture at a initial inoculum density of 1.0E06 cells/mL in 250 mL flasks with 100 mL of TAP media. Cells were allowed to grow in orbital shaker under constant light for 72 hours. After 72 hours, experiment was terminated and cells were harvested via centrifugation.

Treatment ID:

TR000659

Treatment Summary:

Cells were treated either with compounds (5 uM) final concentration in DMSO or DMSO alone (in case of control). Mid-log phase cells were used as starter culture at a initial inoculum density of 1.0E06 cells/mL in 250 mL flasks with 100 mL of TAP media. Cells were allowed to grow in orbital shaker under constant light for 72 hours. After 72 hours, experiment was terminated and cells were harvested via centrifugation.

Sample Preparation:

Sampleprep ID:	SP000652
Sampleprep Summary:	Harvested cells were flash frozen in liquid N and freeze dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min. One milliliter of extraction buffer containing MeOH:CHCl3:H2O (5:2:2; v/v/v; pre-cooled at -20 °C) was added and vortexed for 5 min. Ribitol (0.2 mg/mL in water; 10 µL) was spiked in the extraction buffer as internal standard in order to identify potential chromatographic errors. The homogenized material was centrifuged at 14000 rpm for 5 min and the supernatant was transferred to new tubes. 400 µL of pure water was added to the supernatant, samples were vortexed and centrifuged at 14,000 rpm for 5 min. The upper polar phase was transferred to new tubes for GC-MS analysis. An aliquot of 300 µL was dried out in vacuum concentrator without heating. To the dried material, 10 µL methoxyamine HCL in 100% pyridine (40 mg/mL) was added and shaken at 30 °C for 90 minutes and subsequently 90 µL of MSTFA 1% TMCS was added for trimethylsilylation of acidic protons and shaken at 37 °C for 30 minutes. The reaction mixture was transferred to GCvials with glass microinserts and closed by crimp caps. GC-MS data acquisition was performed as per previously published report (Wase et al., 2014)

Sampleprep ID:

SP000652

Sampleprep Summary:

Harvested cells were flash frozen in liquid N and freeze dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min. One milliliter of extraction buffer containing MeOH:CHCl3:H2O (5:2:2; v/v/v; pre-cooled at -20 °C) was added and vortexed for 5 min. Ribitol (0.2 mg/mL in water; 10 µL) was spiked in the extraction buffer as internal standard in order to identify potential chromatographic errors. The homogenized material was centrifuged at 14000 rpm for 5 min and the supernatant was transferred to new tubes. 400 µL of pure water was added to the supernatant, samples were vortexed and centrifuged at 14,000 rpm for 5 min. The upper polar phase was transferred to new tubes for GC-MS analysis. An aliquot of 300 µL was dried out in vacuum concentrator without heating. To the dried material, 10 µL methoxyamine HCL in 100% pyridine (40 mg/mL) was added and shaken at 30 °C for 90 minutes and subsequently 90 µL of MSTFA 1% TMCS was added for trimethylsilylation of acidic protons and shaken at 37 °C for 30 minutes. The reaction mixture was transferred to GCvials with glass microinserts and closed by crimp caps. GC-MS data acquisition was performed as per previously published report (Wase et al., 2014)

Combined analysis:

Analysis ID	AN000954
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 6890N
Column	Agilent DB-5MS UI Capillary column
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5973
Ion Mode	POSITIVE
Units	peak area

Chromatography:

Chromatography ID:	CH000679
Instrument Name:	Agilent 6890N
Column Name:	Agilent DB-5MS UI Capillary column
Internal Standard:	Ribitol
Sample Injection:	1 uL
Chromatography Type:	GC

MS:

MS ID:	MS000849
Analysis ID:	AN000954
Instrument Name:	Agilent 5973
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE