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MB Sample ID: SA354399
Local Sample ID: | E_L4B |
Subject ID: | SU003390 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 12-16 weeks |
Weight Or Weight Range: | 20-30g |
Gender: | Male |
Animal Animal Supplier: | Phgdh floxed/floxed from the Shigeki Furuya Lab; E2a-Cre mice from JAX |
Animal Housing: | 5/cage |
Animal Light Cycle: | 12h dark/12h light |
Animal Feed: | Special diets; see group labeling |
Animal Water: | Ad libitum |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003390 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 12-16 weeks |
Weight Or Weight Range: | 20-30g |
Gender: | Male |
Animal Animal Supplier: | Phgdh floxed/floxed from the Shigeki Furuya Lab; E2a-Cre mice from JAX |
Animal Housing: | 5/cage |
Animal Light Cycle: | 12h dark/12h light |
Animal Feed: | Special diets; see group labeling |
Animal Water: | Ad libitum |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
E_L4B | SA354399 | FL041358 | PHGDH+/- | Genotype |
E_L4B | SA354399 | FL041358 | Serine/glycine-depleted diet | Treatment |
E_L4B | SA354399 | FL041358 | Mouse back of eye | Sample source |
Collection:
Collection ID: | CO003383 |
Collection Summary: | Tissues were collected using Wollenberger clamps pre-cooled to the temperature of liquid nitrogen and stored at -80℃ until analysis |
Sample Type: | Back of eye (choroid/RPE) |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR003399 |
Treatment Summary: | In this cohort, there were wildtype (WT) and PHGDH heterozygous (PHGDH +/-) mice that were put on either a control amino acid or serine/glycine-depleted diet. |
Sample Preparation:
Sampleprep ID: | SP003397 |
Sampleprep Summary: | For sphingolipid extraction from retina and choroid/RPE, frozen tissue was homogenized with a ball mill (Retsch Mixer Mill MM 400) at 30 Hz for 3 min in 500 μl of −20°C methanol, 400 μL of ice-cold saline, and 100 μL of ice-cold MilliQ water, spiked with deuterated internal standards as described earlier. The mixture was then transferred into a 2-ml Eppendorf tube containing 1 ml of chloroform. The tubes were vortexed for 5 min and centrifuged at 16,000g at 4°C for 5 min. The lower organic phase was collected, and 2 μL of formic acid was added to the remaining polar phase, which was re-extracted with 1 ml of chloroform. Combined organic phases were dried and resuspended in 50 μL of 0.2% formic acid and 1 mM ammonium formate in methanol. Last, the tubes were sonicated in a bath sonicator for 10 min and spun at 16,000g for 10 min at 4°C. For plasma, 50 μl of plasma was extracted with 500 μl of −20°C methanol, 400 μl of saline, and 100 μl of water spiked with deuterated internal standards. Chloroform (1 mL) was then added to the tubes. The tubes were vortexed for 5 min and centrifuged at 16,000g at 4°C for 5 min. The lower organic phase was collected, and 2 μl of formic acid was added to the remaining polar phase, which was reextracted with 1 mL of chloroform. Combined organic phases were dried and resuspended in 100 μl of 0.2% formic acid and 1 mM ammonium formate in methanol. Next, the tubes were sonicated in a bath sonicator for 10 min and spun at 16,000g for 10 min at 4°C |
Processing Storage Conditions: | Room temperature |
Extract Storage: | 4℃ |
Combined analysis:
Analysis ID | AN005356 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Agilent 6460 |
Column | Peeke Scientific Spectra C8SR (150 x 3.0 mm, 3μm) |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | Agilent 6460 QQQ |
Ion Mode | POSITIVE |
Units | Normalized ion count/mg of protein |
Chromatography:
Chromatography ID: | CH004057 |
Chromatography Summary: | The gradient elution program consisted of the following profile: 0 min, 82% B; 3 min, 82% B; 4 min, 90% B, 18 min, 99% B; 25 min, 99%, 27 min, 82% B, 30 min, 82% B. |
Instrument Name: | Agilent 6460 |
Column Name: | Peeke Scientific Spectra C8SR (150 x 3.0 mm, 3μm) |
Column Temperature: | 40°C |
Flow Gradient: | 0 min, 82% B; 3 min, 82% B; 4 min, 90% B; 18 min, 99% B; 25 min, 99% B; 27 min, 82% B; and 30 min, 82% B |
Flow Rate: | 0.5 mL/min |
Solvent A: | 100% water; 0.2% formic acid; 2 mM ammonium formate |
Solvent B: | 100% methanol; 0.2% formic acid; 1 mM ammonium formate |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS005086 |
Analysis ID: | AN005356 |
Instrument Name: | Agilent 6460 QQQ |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | Software: Agilent MassHunter Sphingolipid species were analyzed by selective reaction monitoring of the transition from precursor to product ions at associated optimized collision energies, and fragmentor voltages are provided elsewhere (https://doi.org/10.1007/978-1-60761-322-0_22). The m/z values of the precursor and product ions are provided in the metabolite metadata section. |
Ion Mode: | POSITIVE |