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MB Sample ID: SA338103
| Local Sample ID: | CD041_001 |
| Subject ID: | SU003237 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003237 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CD041_001 | SA338103 | FL040088 | Mouse Embryonic Stem Cells | Sample source |
| CD041_001 | SA338103 | FL040088 | No Glucose | Treatment |
Collection:
| Collection ID: | CO003230 |
| Collection Summary: | Day 4 gastruloids from 2 plates were collected (approximately 45-48 gastruloids, per treatment, per biological replicate) (N, biological replicate = 4) into ice cold PBS in a clean glass petri dish, washed once and transferred to another glass petri dish on ice containing cold PBS. Gastruloids were collected, briefly centrifuged to settle them down and the PBS was completely removed. Gastruloids were snap-frozen in liquid nitrogen. To count the number of cells in each gastruloid, 5 gastruloids from each batch and each treatment, were collected, trypsinised for 1 min in 50 µl of 0.05% trypsin (25300054, ThermoFisher Scientific) and neutralised using 950 µl of warm N2B27. Cells were immediately counted using the Neubauer haemocytometer chamber. |
| Collection Protocol Filename: | Sample_Collection_CD.pdf |
| Sample Type: | Embryonic cells |
| Storage Conditions: | Described in summary |
Treatment:
| Treatment ID: | TR003246 |
| Treatment Summary: | Mouse embryonic stem cell derived gastruloids were treated with different glucose concentrations (0, 21.25mM and 63.75mM) and 5mM of 2-deoxy-D-Glucose between day 3 and 4 of their developmental time window. |
Sample Preparation:
| Sampleprep ID: | SP003244 |
| Sampleprep Summary: | Sample preparation: Metabolite extraction was performed by addition of 200 µL 80 % methanol (including 2 % (v/v) internal standards) and subsequent homogenization on dry ice via a bead beater (FastPrep-24; MP Biomedicals, CA, USA) at 6.0 m/s (5 x 30 s, 5 min pause time) using 1.0 mm zirconia/glass beads (Biospec Products, OK, USA). After centrifugation for 10 min at 15,000 g and 4 °C with a 5415R microcentrifuge (Eppendorf, Hamburg, Germany), supernatants were transferred and the remaining sample residues were reextracted with 200 µL acetonitrile:methanol:water (2:2:1, v/v) containing 1 % (v/v) formic acid using identical settings for homogenization and centrifugation. Corresponding supernatants of both extraction steps were combined and dried under a stream of nitrogen. Dried samples were reconstituted in 60 µL acetonitrile:methanol:water (2:2:1, v/v), vortexed for 5 min, centrifuged, and transferred to analytical glass vials. The LC-MS/MS analysis was initiated within one hour after the completion of the sample preparation. |
| Sampleprep Protocol Filename: | Sample_Collection_CD.pdf |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | Described in summary |
Combined analysis:
| Analysis ID | AN005114 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Vanquish UHPLC |
| Column | Atlantis Premier BEH Z-HILIC (100 x 2.1 mm, 1.7 um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Fisher Scientific Orbitrap Exploris 240 |
| Ion Mode | NEGATIVE |
| Units | counts per second (cps) |
Chromatography:
| Chromatography ID: | CH003870 |
| Chromatography Summary: | Snap-frozen gastruloids were sent to EMBL-Heidelberg, Germany, for the metabolomics analysis. Reagents: LC-MS grade water, acetonitrile and methanol were obtained from Th. Geyer (Germany). High-purity ammonium acetate, ammonium hydroxide, and formic acid were purchased from Merck (Germany). Stable isotope labelled amino acids (MSK-MET1-1; Cambridge Isotope Laboratories, MA, USA) were used as internal standards for untargeted metabolomics. LC-MS/MS analysis was performed on a Vanquish UHPLC system coupled to an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA) in negative ESI (electrospray ionization) mode. Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC column (Waters, MA, USA; 2.1?mm x 100 mm, 1.7 µm) at a flow rate of 0.25 mL/min. The mobile phase consisted of water:acetonitrile (9:1, v/v; mobile phase A) and acetonitrile:water (9:1, v/v; mobile phase B), which were modified with a total buffer concentration of 10 mM ammonium acetate. The aqueous portion of each mobile phase was adjusted to pH 9.0 via addition of ammonium hydroxide. The following gradient (20 min total run time including re-equilibration) was applied (time[min]/%B): 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95. Column temperature was maintained at 40°C, the autosampler was set to 4°C and sample injection volume was 7 µL. |
| Methods Filename: | LC_MS_MS_Full_Protocol.pdf |
| Instrument Name: | Vanquish UHPLC |
| Column Name: | Atlantis Premier BEH Z-HILIC (100 x 2.1 mm, 1.7 um) |
| Column Temperature: | 40 |
| Flow Gradient: | time [min]/%B - 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95 |
| Flow Rate: | 0.25mL/min |
| Solvent A: | water:acetonitrile 9:1, v/v; 10mM ammonium acetate |
| Solvent B: | acetonitrile:water 9:1, v/v; 10mM ammonium acetate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004851 |
| Analysis ID: | AN005114 |
| Instrument Name: | Thermo Fisher Scientific Orbitrap Exploris 240 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Analytes were recorded via a full scan with a mass resolving power of 120,000 over a mass range from 60 – 900 m/z (scan time: 100 ms, RF lens: 70%). To obtain MS/MS fragment spectra, data-dependant acquisition was carried out (resolving power: 15,000; scan time: 22 ms; stepped collision energies [%]: 30/50/70; cycle time: 900 ms). Ion source parameters were set to the following values: spray voltage: 4100 V (positive mode) / -3500 V (negative mode), sheath gas: 30 psi, auxiliary gas: 5 psi, sweep gas: 0 psi, ion transfer tube temperature: 350°C, vaporizer temperature: 300°C. All experimental samples were measured in a randomized manner. Pooled quality control (QC) samples were prepared by mixing equal aliquots from each processed sample. Multiple QCs were injected at the beginning of the analysis in order to equilibrate the analytical system. A QC sample was analyzed after every 5th experimental sample to monitor instrument performance throughout the sequence. For determination of background signals and subsequent background subtraction, an additional processed blank sample was recorded. Data was processed using MS-DIAL and raw peak intensities for relative metabolite quantification. Feature identification was based on accurate mass, isotope pattern, MS/MS fragment scoring and retention time matching to an inhouse library. (Data was acquired on both the positive and the negative modes and the respective raw files are deposited. However, we have processed the data acquired only on the negative mode.) |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | LC_MS_MS_Full_Protocol.pdf |
