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MB Sample ID: SA314550

Local Sample ID:AK15-D8-S-06
Subject ID:SU002991
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

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Subject:

Subject ID:SU002991
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
AK15-D8-S-06SA314550FL037457AkitaGenotype
AK15-D8-S-06SA314550FL0374574 hoursU13C Glucose Labelling Timepoint

Collection:

Collection ID:CO002984
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:TR003000
Treatment Summary:Pregnant mice were placed under a maintained plane of isoflurane anesthesia (2.5%). Retro-orbital blood draw for time zero was performed followed by opening a small incision to the lower abdomen of the pregnant mouse to expose a single conceptus for fetal tissue harvesting. Immediately after the first blood draw and fetus collection, a tail vein catheter was placed and bolus (4 µL/gBW) was administered followed by continuous infusion (0.085ul/gBW/min). A single conceptus, accessed via the initial small incision, was harvested at multiple time points during infusion of the nutrient tracer (13C glucose). Retro-orbital blood draws (<20 ul) were taken throughout the infusion to monitor tracer enrichment in maternal blood. Timepoints for harvesting after time zero were: 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours.

Sample Preparation:

Sampleprep ID:SP002997
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 AN004715 AN004716
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:CH003551
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:MS004461
Analysis ID:AN004715
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:MS004462
Analysis ID:AN004716
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
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