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MB Sample ID: SA205784

Local Sample ID:shctrl HG-2
Subject ID:SU002230
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

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Subject:

Subject ID:SU002230
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
shctrl HG-2SA205784FL025252ControlGenotype

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
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