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MB Sample ID: SA246079
| Local Sample ID: | 01_WT_1 |
| Subject ID: | SU002548 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
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Subject:
| Subject ID: | SU002548 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 01_WT_1 | SA246079 | FL030879 | Wild-type | Genotype |
| 01_WT_1 | SA246079 | FL030879 | none | Treatment |
Collection:
| Collection ID: | CO002541 |
| Collection Summary: | PTCs were isolated as previously described(PMID: 32484794). Briefly, kidneys were excised in ice-cold HBSS buffer. The kidneys were sliced and chopped into pieces (~0.5–1 mm) on ice using a surgical scalpel. The chopped kidneys were transferred into an HBSS solution containing 1 μg/μL collagenase/dispase (Sigma Aldrich, Cat. # 10269638001) and incubated for 25 minutes at 370 C. The cells were filtered through a 40-μm nylon cell strainer (Corning, Cat. # 431750) and washed twice with cold HBSS. For PTC isolation, the cells were stained with PE-conjugated antiCD133/prominin-1 antibody (Invitrogen, Cat. # 12-1331-82) according to the manufacturer's instructions. PE+ cells were isolated by Aria III flow cytometry-based cell sorting. |
| Sample Type: | Proximal tubular cells |
Treatment:
| Treatment ID: | TR002560 |
| Treatment Summary: | A mixture of six labeled internal standards was added to the extraction solution for quality control (13C6-Glucose, 13C5-Glutamine, 13C5-Glutamate, 13C1-Alanine, 13C3-Pyruvate and 13C3-Lactate). The exact volume at each tube was adjusted according to tissue weight (average volume of 200 µl). The samples were homogenized using Precellys 24 tissue homogenizer (Bertin Technologies) precooled to 4°C (3 cycles × 30 s at 6000 rpm, with a 30 s gap between each cycle) and later centrifuged at 18,000 g for 15 min at 4°C. The supernatants were transferred into HPLC glass vials and kept at −80°C prior to LC-MS analysis. |
Sample Preparation:
| Sampleprep ID: | SP002554 |
| Sampleprep Summary: | The samples were homogenized using Precellys 24 tissue homogenizer (Bertin Technologies) precooled to 4°C (3 cycles × 30 s at 6000 rpm, with a 30 s gap between each cycle) and later centrifuged at 18,000 g for 15 min at 4°C. The supernatants were transferred into HPLC glass vials and kept at −80°C prior to LC-MS analysis. |
Combined analysis:
| Analysis ID | AN004010 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Merck 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 | concentration |
Chromatography:
| Chromatography ID: | CH002961 |
| Chromatography Summary: | UPLC setup consisted a ZIC-pHILIC column (SeQuant; 150 mm × 2.1 mm, 5 μm; Merck). 5 µl of cells or kidney extracts were injected using an autosampler. Compounds were separated using a 15 minutes gradient, starting at 20% aqueous (20 mM ammonium carbonate adjusted to pH 9.2 with 0.1% of 25% ammonium hydroxide) and 80% organic (acetonitrile), terminated with 20% acetonitrile. Flow rate and column temperature were kept at 0.2 ml/min and 45°C, respectively for a total run time of 27 min |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 45 |
| Flow Gradient: | 15 min terminated with 20% acetonitrile |
| Flow Rate: | 0.2 ml/min |
| Solvent A: | 20% water/80% acetonitrile; 20 mM ammonium carbonate; 0.1% of 25% ammonium hydroxide (adjusted to pH 9.2) |
| Solvent B: | 80%water/20% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003757 |
| Analysis ID: | AN004010 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Briefly, Dionex Ultimate ultra-high-performance liquid chromatography (UPLC) system coupled to Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific) was used. The resolution was set to 35,000 at 200 mass/charge ratio (m/z) with electrospray ionization and polarity switching mode to enable both positive and negative ions across a mass range of 67–1000 m/z. |
| Ion Mode: | UNSPECIFIED |
