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MB Sample ID: SA205786
Local Sample ID: | shctrl HG-3 |
Subject ID: | SU002230 |
Subject Type: | Cultured cells |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002230 |
Subject Type: | Cultured cells |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
shctrl HG-3 | SA205786 | FL025252 | Control | Genotype |
Collection:
Collection ID: | CO002223 |
Collection Summary: | Conditionally immortalized mouse podocytes were cultured as previously reported.25, 35 Podocytes were treated with 20μM of DL-α-palmitin (Sigma-Aldrich), 1-O-Hexadecyl-rac-glycerol (16:0-AG, Santa Cruz) ,1-O-Octadecyl-rac-glycerol (18:0-AG, Sigma-Aldrich) or 0.05% ethanol (vehicle) for 48 hours. An engineered miR-30 based ChREBP knockdown (KD) construct was assembled by PCR using previously validated ChREBP shRNA clone,34 followed by an optimized miR-E backbone. Stable podocyte cell line with ChREBP KD were generated by transfection into constructs together with PiggyBac transposase. After selection with 0.5 μg/ml of blasticidin alone or in combination with 1μg/ml puromycin, GFP-positive cells were sorted by FACS, and the top 30% population were collected. |
Sample Type: | Epithelial cells |
Treatment:
Treatment ID: | TR002242 |
Treatment Summary: | An engineered miR-30 based ChREBP knockdown (KD) construct was assembled by PCR using previously validated ChREBP shRNA clone,34 followed by an optimized miR-E backbone. Stable podocyte cell line with ChREBP KD were generated by transfection into constructs together with PiggyBac transposase. After selection with 0.5 μg/ml of blasticidin alone or in combination with 1μg/ml puromycin, GFP-positive cells were sorted by FACS, and the top 30% population were collected. |
Sample Preparation:
Sampleprep ID: | SP002236 |
Sampleprep Summary: | Mitochondria were isolated from mouse podocytes using Mitochondria Isolation Kit (Thermo Fisher). Mitochondria were pelleted by centrifugation at 3000x g for 15 minutes at 4°C, flash-frozen and stored at -80°C until use. 200 μL of ethanol containing 1% 10 mM butylated hydroxytoluene in methanol, and 2% Avanti SPLASH® LIPIDOMIX® Mass Spec Standards, pre-cooled to -80°C, was added to each mitochondria sample. The tubes were vortexed for 10 min, placed on ice for 10 min, then centrifuged at 4°C for 10 min at 17,000 x g. The supernatants were then collected for LC-MS analysis. |
Combined analysis:
Analysis ID | AN003511 | AN003512 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Thermo Vanquish | Thermo Vanquish |
Column | Thermo Accucore C18 (100 x 2.1mm,2.6um) | Thermo Accucore C18 (100 x 2.1mm,2.6um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Fusion Orbitrap | Thermo Fusion Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | AUC/ngDNA | AUC/ngDNA |
Chromatography:
Chromatography ID: | CH002592 |
Chromatography Summary: | Mobile phase A (MPA) was 40:60 acetonitrile:0.1% formic acid in 10 mM ammonium formate. Mobile phase B (MPB) was 90:9:1 isopropanol: acetonitrile: 0.1% formic acid in 10 mM ammonium formate. The chromatographic method included a Thermo Fisher Scientific Accucore C30 column (2.6 μm, 150 x 2.1 mm) maintained at 40°C, a mobile phase flowrate of 0.200 mL/min, and a gradient elution program as follows: 0-3 min, 30% MPB; 3-13 min, 30-43% MPB; 13.1-33 min, 50-70% MPB; 33-48 min, 70-99% MPB; 48-55 min, 99% MPB; 55.1-60 min, 30% MPB. |
Instrument Name: | Thermo Vanquish |
Column Name: | Thermo Accucore C18 (100 x 2.1mm,2.6um) |
Column Temperature: | 40 |
Flow Gradient: | 0-3 min, 30% B; 3-13 min, 30-43% B; 13.1-33 min, 50-70% B; 33-48 min, 70-99% B; 48-55 min, 99% B; 55.1-60 min, 30% B |
Flow Rate: | 0.200 mL/min |
Solvent A: | 40% acetonitrile/60% water; 0.1% formic acid; 10 mM ammonium formate |
Solvent B: | 90% isopropanol/9% acetonitrile/1% water; 0.1% formic acid; 10 mM ammonium formate |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS003269 |
Analysis ID: | AN003511 |
Instrument Name: | Thermo Fusion Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | A Thermo Fisher Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer with heated electrospray ionization source was operated in data- dependent acquisition mode, in positive ionization mode, with scan ranges of 150-827 and 825-1500 m/z. An Orbitrap resolution of 120,000 (FWHM) was used for MS1 acquisition and spray voltages of 3.6kV and -2.9kV were used for positive and negative ionization modes, respectively. For MS2 and MS3 fragmentation a hybridized HCD/CID approach was used. Each sample was analyzed using 4 x 10 µL injections making use of the two scan ranges, in both ionization modes. Data were analyzed using Thermo Scientific LipidSearch software (version 5.0.63) and R scripts written in house. The intensity of each peak was normalized to total lipid signal and to the internal standard. |
Ion Mode: | POSITIVE |
MS ID: | MS003270 |
Analysis ID: | AN003512 |
Instrument Name: | Thermo Fusion Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | A Thermo Fisher Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer with heated electrospray ionization source was operated in data- dependent acquisition mode, in negative ionization mode, with scan ranges of 150-827 and 825-1500 m/z. An Orbitrap resolution of 120,000 (FWHM) was used for MS1 acquisition and spray voltages of 3.6kV and -2.9kV were used for positive and negative ionization modes, respectively. For MS2 and MS3 fragmentation a hybridized HCD/CID approach was used. Each sample was analyzed using 4 x 10 µL injections making use of the two scan ranges, in both ionization modes. Data were analyzed using Thermo Scientific LipidSearch software (version 5.0.63) and R scripts written in house. The intensity of each peak was normalized to total lipid signal and to the internal standard. |
Ion Mode: | NEGATIVE |