Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA035039

Local Sample ID:	784_B_3
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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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)