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MB Sample ID: SA169164
| Local Sample ID: | RBP42_KD_4 |
| Subject ID: | SU001899 |
| Subject Type: | Cultured cells |
| Subject Species: | Trypanosoma brucei brucei |
| Taxonomy ID: | 5702 |
| Genotype Strain: | 427 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001899 |
| Subject Type: | Cultured cells |
| Subject Species: | Trypanosoma brucei brucei |
| Taxonomy ID: | 5702 |
| Genotype Strain: | 427 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| RBP42_KD_4 | SA169164 | FL020188 | Knockdown | Treatment |
Collection:
| Collection ID: | CO001892 |
| Collection Summary: | T. brucei Lister 427 bloodstream form RBP42 conditional knockdown cell line (RBP42Ty1) was grown in HMI-9 medium supplemented with 10% fetal bovine serum and 10% serum plus at 37C in a humidified incubator containing 5% CO2. Cultures were seeded at 1 x 10^5 cells/ml, passaged every 2-3 days and grown to a maximum of 1x10^6 cells per ml. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR001912 |
| Treatment Summary: | For metabolomic analysis, RBP42Ty1 cells were collected before and after two days of RBP42 depletion. Cells (2 x 10^7 cells/assay) were harvested and metabolites were extracted. |
Sample Preparation:
| Sampleprep ID: | SP001905 |
| Sampleprep Summary: | Metabolites were extracted with 1 ml of 40:40:20 mixture of methanol:acetonitrile:water plus 0.5% (V/V) formic acid on ice for 5 min. Following neutralization of formic acid by addition of 50 ul of 15% (m/V) NH4HCO3, cleared extracts were collected by centrifugation at 15000g for 10 min and stored at -80C. |
Combined analysis:
| Analysis ID | AN002958 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | arbitrary |
Chromatography:
| Chromatography ID: | CH002191 |
| Chromatography Summary: | HILIC separation was performed on a Vanquish Horizon UHPLC system (Thermo Fisher Scientific, Waltham, MA) with an XBridge BEH Amide column (150 mm × 2.1 mm, 2.5 μm particle size, Waters, Milford, MA) using a gradient of solvent A (95%:5% H2O:acetonitrile with 20 mM acetic acid, 40 mM ammonium hydroxide, pH 9.4) and solvent B (20%:80% H2O:acetonitrile with 20 mM acetic acid, 40 mM ammonium hydroxide, pH 9.4). The gradient was 0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B; 10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45% B; 16.5 min, 100% B; and 22 min, 100% B. The flow rate was 300 μl/min. The injection volume was 5 μL, and the column temperature was set to 25°C. The autosampler temperature was set to 4°C, and the injection volume was 5 µL. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Column Temperature: | 25 |
| Flow Gradient: | 0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B; 10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45% B; 16.5 min, 100% B; and 22 min, 100% B. |
| Flow Rate: | 300 µl/min |
| Solvent A: | 95% water/5% acetonitrile; 20 mM acetic acid; 40 mM ammonium hydroxide, pH 9.4 |
| Solvent B: | 20% water/80% acetonitrile; 20 mM acetic acid, 40 mM ammonium hydroxide, pH 9.4 |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002748 |
| Analysis ID: | AN002958 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Each sample was analyzed with a nominal resolution of 70,000. The automatic gain control target was 3×106. The maximum injection time was 50 ms. Scan range was 75-1,000. The targeted metabolite data analysis was performed in MAVEN. The compound identification was based on the accurate mass and the retention time learned from in-house chemical collection |
| Ion Mode: | NEGATIVE |
