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MB Sample ID: SA313224
| Local Sample ID: | heart-AK-E12-D-02 |
| Subject ID: | SU002983 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002983 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| heart-AK-E12-D-02 | SA313224 | FL037415 | AK | Genotype |
| heart-AK-E12-D-02 | SA313224 | FL037415 | heart | Tissue |
Collection:
| Collection ID: | CO002976 |
| Collection Summary: | Healthy wildtype and Akita dams were set up for mating. The following morning, females displaying vaginal plugs were identified as pregnant, recorded as embryonic day (E) 0.5 and moved to a new cage until the appropriate embryonic day to be interrogated. |
| Sample Type: | Embryo |
Treatment:
| Treatment ID: | TR002992 |
| Treatment Summary: | Labeled glucose solution was prepared at a concentration of 100 mg/mL in filtered 0.9% sodium chloride solution. After overnight fasting (from 18:00 the day before), infusions took place around between 09:00 and 10:00 for all pregnant dams. Mice were anesthetized using isoflurane gas at 5% and placed on a warm pad. Mice were then kept under 2.5% isoflurane for the duration of infusion. For catheter placement, a 28-gauge insulin syringe needle was connected via polyethylene tubing (PE-10) to a syringe (containing glucose solution) placed on an infusion pump (Harvard Apparatus). At the start of the infusion, the 28-gauge needle was inserted into the tail vein. A glucose bolus of 4 µL/gBW was administered. Right after bolus administration, infusion rate was set at a continuous 0.085ul/gBW for a total infusion time of 3 hours. |
Sample Preparation:
| Sampleprep ID: | SP002989 |
| Sampleprep Summary: | Fetal tissue extraction: Following infusion, mice were euthanized and blood was collected via heart puncture. Fetal tissues (placenta, brain, liver, and heart) were dissected in ice-cold sterile PBS. Immediately after dissection, weight was recorded and fetal tissue was placed in a pre-filled bead mill tube containing metal beads and 500 µL of methanol:water (80:20) solution kept cold on dry ice. Fetal tissues were homogenized using a Fisherbrand™ Bead Mill Homogenizer. Samples were spun twice at >17,000 g (4 °C) to remove precipitated cell material (protein/DNA). Supernatants were collected, transferred to a clean tube, and evaporated using a Nitrogen evaporator (Organomation). Evaporated samples were stored at -80 °C. Pellets containing protein/DNA were dried on a heat block (55 °C) and stored at -80 °C. Serum extraction: Collected blood was centrifuged at 5,000g to collect serum. Serum was snap frozen in liquid nitrogen and stored until extraction. For metabolite extraction 5 µL serum was mixed with 500 µL 100% MeOH (-80 °C). Samples were centrifuged for 10 min at >17,000 g (4 °C) and 450 µL of each sample evaporated using a Nitrogen evaporator (Organomation). Evaporated samples were stored at -80 °C. |
| Processing Storage Conditions: | On ice |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN004705 | AN004706 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | SeQuant ZIC-HILIC (150 x 2.1mm,5um) | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak Area | Peak Area |
Chromatography:
| Chromatography ID: | CH003543 |
| Chromatography Summary: | Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile (ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 min at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials. 10 µL of these metabolite solutions were injected per analysis. Samples were run on a Vanquish (Thermo Scientific) UHPLC system with mobile phase A (20mM ammonium carbonate, pH 9.7) and mobile phase B (100% ACN) at a flow rate of 150 µL/min on a SeQuant ZIC-pHILIC Polymeric column (2.1 × 150 mm 5 μm, EMD Millipore) at 35°C. Separation was achieved with a linear gradient from 20% A to 80% A in 20 min followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A was then held from 20.5 min to 28 min. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| Column Temperature: | 35°C |
| Flow Gradient: | Separation was achieved with a linear gradient from 20% A to 80% A in 20 min followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A was then held from 20.5 min to 28 min. |
| Flow Rate: | 150 µL/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate, pH 9.7 |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004451 |
| Analysis ID: | AN004705 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The UHPLC was coupled to a Q-Exactive (Thermo Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range = (70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard. These “.mzXML” files were imported into the MZmine 2 software package. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor 2 to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004452 |
| Analysis ID: | AN004706 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The UHPLC was coupled to a Q-Exactive (Thermo Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range = (70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard. These “.mzXML” files were imported into the MZmine 2 software package. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor 2 to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well. |
| Ion Mode: | NEGATIVE |
