Return to study ST002184 main page
MB Sample ID: SA209986
| Local Sample ID: | CM02-068 |
| Subject ID: | SU002270 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002270 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CM02-068 | SA209986 | FL025605 | Das2_VillinCreERT2_wt/wt | Genotype |
| CM02-068 | SA209986 | FL025605 | intestinal epithelial cells | Tissue type |
Collection:
| Collection ID: | CO002263 |
| Collection Summary: | Small intestines were dissected and washed with PBS. Small pieces of approximately ~0.5 cm were isolated from proximal (post stomach) small intestine and snap frozen on dry ice and stored at -80°C until further processing. The remaining small intestinal tissue was washed in PBS to remove faeces and cut longitudinally. Intestinal epithelial cells were isolated by sequential incubation of intestinal tissue in pre-heated 1 mM dithiothreitol and 1.5 mM EDTA solutions at 37oC while shaking. Intestinal epithelial cell pellet was frozen at -80°C until further processing. Intestinal epithelial cell (IEC) pellet was frozen at -80oC for further processing. |
| Sample Type: | Intestine |
Treatment:
| Treatment ID: | TR002282 |
| Treatment Summary: | VillinCreERT2 recombinase activity was induced by five consecutive daily intraperitoneal administrations of 1 mg tamoxifen dissolved in corn oil/DMSO. Small intestine tissue and intestinal epithelial cells were harvested 8 days post last tamoxifen treatment. |
Sample Preparation:
| Sampleprep ID: | SP002276 |
| Sampleprep Summary: | Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% water, 5uM valine-d8 as internal standard) was added to (10-20mg) frozen small intestine tissue samples at an extraction ratio of 25ul/mg on dry ice. Samples were then homogenized using a Precellys 24 tissue homogenizer (Bertin technologies). The resulting sample suspension was vortexed, mixed at 4oC in a Thermomixer for 15 min at 1,500 rpm and then centrifuged at 16,000 x g for 20 min at 4oC. The supernatant was collected for LC-MS analysis. |
Combined analysis:
| Analysis ID | AN003577 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002645 |
| Chromatography Summary: | LC-MS chromatographic separation of metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40°C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B. Samples were randomized and analysed with LC–MS in a blinded manner with an injection volume of 5 µl. Pooled samples were generated from an equal mixture of all individual samples and analysed interspersed at regular intervals within sample sequence as a quality control. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 40 |
| Flow Gradient: | 0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B. |
| Flow Rate: | 0.200 mL/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003334 |
| Analysis ID: | AN003577 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Metabolites were measured with a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass spectrometer (HRMS) coupled to a Dionex Ultimate 3000 UHPLC. The mass spectrometer was operated in full-scan, polarity-switching mode, with the spray voltage set to +4.5 kV/-3.5 kV, ion transfer tube temperature set to 320 °C, the vaporizer temperature set to 280 °C, the sheath gas flow set to 55 units, the auxiliary gas flow set to 15 units, and the sweep gas flow set to 0 unit. HRMS data acquisition was performed in a range of m/z = 70–900, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time (Max IT) at 120 ms. Chromatogram review and peak area integration were performed using the Thermo Fisher software Tracefinder (v.5.0). Metabolite identities were confirmed using two parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. |
| Ion Mode: | UNSPECIFIED |
