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MB Sample ID: SA341050

Local Sample ID:STD6_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 IDMB Sample IDFactor Level IDLevel ValueFactor Name
STD6_aSA341050FL040349Calibration CurveSample source
STD6_aSA341050FL040349N/AGenotype

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
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