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MB Sample ID: SA100665
| Local Sample ID: | 2019_09_13_RPIP15_H1-M3_1 |
| Subject ID: | SU001451 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001451 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 2019_09_13_RPIP15_H1-M3_1 | SA100665 | FL014179 | H1 | Cell line |
| 2019_09_13_RPIP15_H1-M3_1 | SA100665 | FL014179 | day 3 | Status |
Collection:
| Collection ID: | CO001446 |
| Collection Summary: | Extracellular metabolite dynamics was analyzed during primed to naïve conversion in the bioreactor. For this, the media of H1 hESCs in the bioreactor was gradually switched from mTESR1 to RSeT media within 6 days of culture (D2 to D6) and the media was collected every day for the analysis. For H9 hESCs, the extracellular metabolites were analyzed in the spent media of primed and established naïve (P5) hPSCs in the bioreactor, and the media of each primed or naïve hPSCs was collected at day four of bioreactor culture. |
| Sample Type: | Stem cells |
Treatment:
| Treatment ID: | TR001466 |
| Treatment Summary: | For H1 hESCs, the extracellular metabolite dynamics was analyzed during primed to naïve conversion in the bioreactor. For this, the media of H1 hESCs in the bioreactor was gradually switched from mTESR1 to RSeT media within 6 days of culture (D2 to D6) and the media was collected every day for the analysis. For H9 hESCs, the extracellular metabolites were analyzed in the spent media of primed and established naïve (P5) hPSCs in the bioreactor, and the media of each primed or naïve hPSCs was collected at day four of bioreactor culture. |
Sample Preparation:
| Sampleprep ID: | SP001459 |
| Sampleprep Summary: | The metabolite extraction was started using LC-MS or HPLC grade methanol (Sigma-Aldrich, 1.06035). Briefly, a 950 μL of pre-chilled 50% MeOH/H2O was added to a 50 μL of the bioreactor-collected media (making a D20 dilution), and the samples were incubated on ice for 30 min to allow for full extraction. Macromolecules were then pelleted by centrifugation at max speed (~18,000-21,000 g) for 10 mins in a bench top centrifuge (preferably chilled) to extract the supernatant. Further, the extracted samples were stored in -80 prior to running HPLC-MS. |
| Processing Storage Conditions: | Room temperature |
| Extraction Method: | 50% Methanol in water |
| Extract Storage: | On ice |
Combined analysis:
| Analysis ID | AN002297 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Agilent Zorbax RRHT SB-C18 (50 x 2.1 mm,1.8um) |
| MS Type | ESI |
| MS instrument type | Single quadrupole |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE |
| Units | relative signal intensity compared to media only |
Chromatography:
| Chromatography ID: | CH001687 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Agilent Zorbax RRHT SB-C18 (50 x 2.1 mm,1.8um) |
| Flow Rate: | 0.6 ml/min |
| Solvent A: | 97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002140 |
| Analysis ID: | AN002297 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Single quadrupole |
| MS Type: | ESI |
| MS Comments: | The mass spectrometer was run in negative full scan mode at a resolution of 140,000 scanning from 50-750m/z. Data was processed using MAVEN. |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 275 |
| Spray Voltage: | 2.50 kV |
| Analysis Protocol File: | MS_Metabolomics.pdf |
