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MB Sample ID: SA341025
Local Sample ID: | STD14_a |
Subject ID: | SU003271 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
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Subject:
Subject ID: | SU003271 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
STD14_a | SA341025 | FL040349 | Calibration Curve | Sample source |
STD14_a | SA341025 | FL040349 | N/A | Genotype |
Collection:
Collection ID: | CO003264 |
Collection Summary: | 8-10 weeks old C57BL/6J female mice were euthanized with 75 µl of a 60 mg ml-1 Dolethal solution (pentobarbital sodium, Vetoquinol). Subsequently, sgNT and sgCda primary tumors were collected by surgical resection, washed with blood bank saline and dried from liquid excess. Tissues were then placed in a home-made filtered centrifugation tube supplemented with a 20 µm nylon mesh filter (Repligen) and centrifuged at 400 x g at 4 °C for 10 minutes. Between 1-14 µl of tumor interstitial fluid were collected and stored on dry ice. Interstitial fluid volume was used to determine the metabolites concentration measured by mass spectrometry. |
Sample Type: | Interstitial fluid |
Treatment:
Treatment ID: | TR003280 |
Treatment Summary: | KPC FC1245 cells were genetically engineered using doxycycline inducible CRISPR/Cas9 system platform to target specifically cytidine deaminase (sgCda). sgCda and the control sgNT were then orthotopically injected in the head of the pancreas of 8-10 weeks old C57BL/6J female mice. When the tumor weight reached approximately 0.2-0.4g, mice were sacrificed and tumor interstitial fluid was collected |
Sample Preparation:
Sampleprep ID: | SP003278 |
Sampleprep Summary: | Standard curves for Glucose (Sigma-Aldrich, G7021), UDP (Sigma-Aldrich, 94330), UTP (Jena Bioscience, NU-1024S), cytidine (Sigma-Aldrich, C4654), uridine (Sigma-Aldrich, U3003) and glutamine (Gibco, 25030-34) were used to calculate the concentration of these metabolites in the samples. The standard curve was prepared with a dilution series starting from 5 mM Glucose and Glutamine, 1 mM Cytidine, Uridine, UDP and UTP. Specific dilutions are indicated in the Study Design table. Metabolites and calibration curves were extracted simultaneously by addition of 800 μl of MS-grade methanol-water buffer (MeOH:H2O, 5:3, v/v) containing the internal standards glutaric acid (5 μg ml-1, Sigma-Aldrich, G3407) and 13C6-Glucose (30 μg ml-1, Cambridge Isotope Laboratories, Inc., CLM-1396), followed by 500 μl of chloroform. Samples were then vortexed and centrifuged (4 °C for 10 min each). The polar (upper) phases were collected, divided into two equal parts for gas chromatography (GC) and liquid chromatography (LC) mass spectrometry analysis, and dried using a vacuum concentrator. The dried metabolite extracts were stored at -80 °C until analysis. |
Combined analysis:
Analysis ID | AN005174 | AN005175 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | GC |
Chromatography system | Thermo Dionex Ultimate 3000 | Agilent 8860 |
Column | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) | Agilent J&W DB-35ms (30 m x 0.25 mm, 0.25 µm) |
MS Type | ESI | EI |
MS instrument type | Orbitrap | Single quadrupole |
MS instrument name | Thermo Q Exactive Orbitrap | Agilent 5977C |
Ion Mode | NEGATIVE | POSITIVE |
Units | Ion Counts (AUC) | Ion Counts (AUC) |
Chromatography:
Chromatography ID: | CH003914 |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
Column Temperature: | 40 |
Flow Gradient: | The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min post injection. A linear gradient to 37% B was carried out until 7 min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min. |
Flow Rate: | 0.25 mL/min |
Solvent A: | 100% water; 10 mM tributyl-amine; 15 mM acetic acid |
Solvent B: | 100% methanol |
Chromatography Type: | Reversed phase |
Chromatography ID: | CH003915 |
Chromatography Summary: | Inlet Temperature: 270; Helium flow rate: 0.88 mL/min; The GC oven was kept at 100°C and for 1 min, increased with a gradient of 2.5°C min-1 to 105°C for 2 min, then ramped with a gradient of 15°C min-1 to 260°, and after that ramped with a gradient of 20°C min-1 up to 300°C for 5 min. |
Instrument Name: | Agilent 8860 |
Column Name: | Agilent J&W DB-35ms (30 m x 0.25 mm, 0.25 µm) |
Column Temperature: | 100-300 |
Flow Gradient: | N/A |
Flow Rate: | N/A |
Solvent A: | N/A |
Solvent B: | N/A |
Chromatography Type: | GC |
MS:
MS ID: | MS004909 |
Analysis ID: | AN005174 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The MS was operated in negative full scan mode (m/z range: 70–900 and 400-500) using a spray voltage of 4.0 kV, capillary temperature of 350 °C, sheath gas at 50.0, auxiliary gas at 10.0. The AGC target was set at 3.0E6 using a resolution if 140000, with a maximum IT fill time of 512 ms. Data was collected and analyzed using the Xcalibur software (Thermo Scientific) considering a 5 ppm error. The chromatographic peaks for UDP, UTP, cytidine, uridine and glutamine were normalized to the internal standard glutaric acid. Tumor interstitial fluid samples were interpolated to the standard curve and normalized for tumor interstitial fluid volume in order to quantify metabolite concentration. |
Ion Mode: | NEGATIVE |
MS ID: | MS004910 |
Analysis ID: | AN005175 |
Instrument Name: | Agilent 5977C |
Instrument Type: | Single quadrupole |
MS Type: | EI |
MS Comments: | 5977C quipped with EI inert source. Mass spectrometry was performed at 70 eV and a mass range of 150-650 atomic mass units was measured. The chromatographic peaks were extracted from raw chromatograms with a custom MATLAB Script. The chromatographic peaks for 12C-Glucose were normalized to the internal standard 13C6-glucose. Tumor interstitial fluid samples were interpolated to the standard curve and normalized for tumor interstitial fluid volume to quantify metabolite concentration. |
Ion Mode: | POSITIVE |