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MB Sample ID: SA028283
| Local Sample ID: | CS44_MUT04_48 |
| Subject ID: | SU000570 |
| Subject Type: | Cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000570 |
| Subject Type: | Cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CS44_MUT04_48 | SA028283 | FL006988 | CS-48h | Time Point |
| CS44_MUT04_48 | SA028283 | FL006988 | 293T HEK cell line | Sample Source |
Collection:
| Collection ID: | CO000564 |
| Collection Summary: | 293T Cells were harvested 24 and 48 hours after transfection by removal of media and rapid quenching with 1.5 mL per 10 cm plate of -80°C methanol. Cells were incubated at -80°C for 15 minutesmin, then scraped off the dish and centrifuged for 5 min at 2,000 x g at 4°C to pellet cellular debris. Pellet was re-extracted by addition of 500 µL of -80°C 80% methanol in water, vortexed, then incubated at 4°C for 15 minutes and centrifuged for 5 minutes at 2,000 x g at 4°C. Peripheral blood mono-nuclear cells were centrifuged at 2,000 x g, freeze media removed and extracted using same procedure as 293T cells. |
| Collection Protocol Filename: | The common feature of IDH1 and IDH2 mutations.pdf |
| Sample Type: | Cells |
| Collection Location: | UC Davis Genome and Biomedical Sciences Facility |
Treatment:
| Treatment ID: | TR000584 |
| Treatment Summary: | 239T cells (ATCC CRL-3216) were grown in DMEM (Invitrogen) with 10% FBS (Hyclone) at -37°C in 10% CO2 and transfected with pcDNA3.1, pcDNA3.1-IDH2WT, pcDNA3.1-IDH2R172K (Invitrogen and Origene) with Lipofectamine 2000 (Invitrogen) according to manufacturer instructions. 6x10^5 cells were seeded in 6 well plates for protocol 1 and 3.5x10^6 cells were seeded in 10 cm plates for protocol 2. Identity of all vectors was confirmed by sequencing and vector integrity with agarose gel electrophoresis.The common feature of IDH1 and IDH2 mutations.pdf |
| Treatment Protocol Filename: | 239T cells (ATCC CRL-3216) were grown in DMEM (Invitrogen) with 10% FBS (Hyclone) at 37°C in 10% CO2 and transfected with pcDNA3.1, pcDNA3.1-IDH2WT, pcDNA3.1-IDH2R172K (Invitrogen and Origene) with Lipofectamine 2000 (Invitrogen) according to manufacturer instructions. |
| Cell Harvesting: | 24-48 hours after transfection |
Sample Preparation:
| Sampleprep ID: | SP000577 |
| Sampleprep Summary: | Supernatants were combined and evaporated to dryness. Samples were then re-suspended in 200 µL LC-MS-grade water. A 2 mL AG-1 X8 100-200 anion exchange resin (Bio-Rad) was washed with 5 column volumes (10 mL) of 3 N HCl followed by transfer of re-suspended extracts to resin column. Metabolites were eluted using 10 mL 3 N HCl. Samples were evaporated to dryness and re-suspended in 100 µL MTBSTFA/ACN (1:1, v/v) and shaken at 60°C for 1 hour. Derivatized extracts were further diluted (1:4) with a MTBSTFA/ACN (1:1, v/v) mixture and transferred to glass vials with micro-inserts and capped immediately. |
| Sampleprep Protocol Filename: | The common feature of IDH1 and IDH2 mutations.pdf |
Combined analysis:
| Analysis ID | AN000836 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890A |
| Column | Agilent HP5-MS (30m × 0.25mm, 0.25 um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent |
| Ion Mode | POSITIVE |
| Units | Counts |
Chromatography:
| Chromatography ID: | CH000597 |
| Methods Filename: | Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf |
| Instrument Name: | Agilent 7890A |
| Column Name: | Agilent HP5-MS (30m × 0.25mm, 0.25 um) |
| Column Temperature: | 250°C |
| Flow Rate: | 1mL/min |
| Sample Injection: | 0.2µl |
| Oven Temperature: | 100°C (3 min), 4°C/min to 230°C (hold 4 min), 30°C/min to 300°C (hold 5 min) |
| Transferline Temperature: | 230°C |
| Randomization Order: | Excel generated |
| Chromatography Type: | GC |
MS:
| MS ID: | MS000737 |
| Analysis ID: | AN000836 |
| Instrument Name: | Agilent |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| Ion Mode: | POSITIVE |
| Ion Source Temperature: | 230°C |
| Ionization Energy: | 70eV |
| Mass Accuracy: | Nominal |
| Scan Range Moverz: | 50-600 Da |
| Scanning Cycle: | 2.71 scans/sec |
| Scanning Range: | 50-600 Da |
