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MB Sample ID: SA037656

Local Sample ID:CSH_PM D3-17 e1_pos mode_MS_07212014.d
Subject ID:SU000679
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57/BL6N
Gender:Male
Animal Housing:SPF facility
Animal Light Cycle:12:12 h light-dark cycle
Animal Feed:Ad libitum
Animal Water:Ad libitum
Species Group:Mammal

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Subject:

Subject ID:SU000679
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57/BL6N
Gender:Male
Animal Housing:SPF facility
Animal Light Cycle:12:12 h light-dark cycle
Animal Feed:Ad libitum
Animal Water:Ad libitum
Species Group:Mammal

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
CSH_PM D3-17 e1_pos mode_MS_07212014.dSA037656FL007968LA-HFDGROUP_DESCRIPTION
CSH_PM D3-17 e1_pos mode_MS_07212014.dSA037656FL00796824 weeksTIME_POINT
CSH_PM D3-17 e1_pos mode_MS_07212014.dSA037656FL007968WT C57-Bl6Genotype

Collection:

Collection ID:CO000673
Collection Summary:Liver tissue from mice on the diets for 24 weeks for metabolomic analysis was collected, rinsed in cold PBS, excess fluid was blotted with a kim-wipe and tissue was immediately snap frozen in liquid nitrogen before storage at -80°C. Blood was collected by cardiac puncture and centrifuged at 9 rcf for 5 min at 4°C. Plasma was stored immediately at -20°C.
Sample Type:Blood

Treatment:

Treatment ID:TR000693
Treatment Summary:Male C57/BL6N mice weaned at 3 weeks of age were randomly assigned to one of the four diets: 1) VIV chow: normal rodent chow, low in fat and high in fiber 2) HFD: 40 kcal% coconut oil 3) LA-HFD: 40 kcal% total fat soybean oil diet (21 kcal% from coconut oil and 19 kcal% from soybean oil) 4) PL-HFD: 40 kcal% total fat Plenish oil diet (21 kcal% from coconut oil and 19 kcal% from Plenish oil)

Sample Preparation:

Sampleprep ID:SP000686
Sampleprep Summary:1) Keep Specimen on Dry Ice 2) Transfer Tissue Contents into a new 1.5mL labeled eppendorf tube; keep on dry ice at all times 3) Add three (3) 3mm metal grinding balls to each sample; store in -80C for 10minutes 4) Homogenize the entire tissue to fine powder using genogrinder; make sure that the metal grinding balls are ice-cold prior to homogenization (step 3) 5) Upon completion of homogenization, keep samples on dry ice 6) Weight out two (2) aliquots: a ~5mg aliquot for CSH_lipidomics and a ~4mg aliquot for Primary Metabolites by GCTOF a. Record the exact weight weighed out for each sample b. Keep all samples on dry ice 7) KEEP remaining tissue specimen (>90mg) for analysis of Oxylipins (store in -80C) 8) Analysis of Primary Metabolites (GCTOFMS) a. Add 1mL of ice-cold “degassed” 3:3:2 ACN/IPA/H2O b. Vortex for 10seconds c. Shake on shaker for 20min at -4C d. Centrifuge the samples for 2min at 14,000 rcf e. Transfer two (2) 500μL aliquots to new 1.5mL eppendorf tubes; one for backup the other to be dried to dryness using the SpeedVac f. IMPORTANT: The precipitated protein will be used for analysis of the proteome, DO NOT DISCARD THESE; Place these in a separate labeled box and store in -20C g. Keep all samples on ice during extraction period h. Dry down one (1) 500μL aliquot to complete dryness i. Perform cleanup on dried aliquot using 500μL of 50/50 v/v ACN/H2O j. Transfer supernatant and dry to completeness k. Submit for Derivatization 9) Analysis of Complex Lipids (LCQTOF) a. Add 225μL of ice-cold “degassed” MeOH containing “ISTD mixture” to homogenized 5mg aliquot b. Vortex for 10 seconds c. Add 750μL of ice-cold “degassed” MTBE containing 22:1 CE ISTD d. Vortex for 10 seconds e. Shake on Orbital Mixer for 6min at 4C f. Add 188μL of room temperature H2O g. Vortex for 20 seconds h. Centrifuge for 2min at 14,000 rcf i. Transfer two (2) aliquots of 350μL of top layer, one for backup stored in -20C, the other for analysis j. Keep bottom layer and store in -20C k. Dry down one (1) 350μL aliquot to dryness using the Speedvac l. Resuspend samples in 108.6μL of 50ng/mL CUDA m. Vortex and sonicate for 5minutes n. Centrifuge for 2min at 14,000 rcf o. Transfer 90μL to an amber vial with micro-insert (non-diluted) p. Transfer 10μL to a new 1.5mL eppendorf tube, dilute 20X with 50ng/mL CUDA in 90:10 MeOH:Toluene (10μL + 190μL CUDA) and transfer 100μL to amber vial with micro-insert (diluted for TGs) i. The dilution is based off previous experiences with liver samples
Sampleprep Protocol Filename:SOP_Sample_preparation_of_blood_plasma_or_serum_samples_for_GCTOF_analysis.pdf

Combined analysis:

Analysis ID AN001001 AN001002
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Agilent 6530 Agilent 6550
Column Waters Acquity CSH C18 (100 x 2.1mm,1.7um) Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Agilent 6530 QTOF Agilent 6550 QTOF
Ion Mode POSITIVE NEGATIVE
Units Counts Counts

Chromatography:

Chromatography ID:CH000718
Methods Filename:Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics_09-27-2013_general.pdf
Instrument Name:Agilent 6530
Column Name:Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Column Pressure:450-850 bar
Column Temperature:65 C
Flow Gradient:15% B to 99% B
Flow Rate:0.6 mL/min
Injection Temperature:4 C
Internal Standard:See data dictionary
Retention Time:See data dictionary
Sample Injection:1.67 uL
Solvent A:60% acetonitrile/40% water; 10mM formic acid; 10mM ammonium formate
Solvent B:90% isopropanol/10% acetonitrile; 10mM formic acid; 10mM ammonium formate
Analytical Time:13 min
Capillary Voltage:3500 eV
Time Program:15 min
Weak Wash Solvent Name:Isopropanol
Strong Wash Solvent Name:Isopropanol
Target Sample Temperature:Autosampler temp 4 C
Randomization Order:Excel generated
Chromatography Type:Reversed phase
  
Chromatography ID:CH000719
Methods Filename:Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics_09-27-2013_general.pdf
Instrument Name:Agilent 6550
Column Name:Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Column Pressure:450-850 bar
Column Temperature:65 C
Flow Gradient:15% B to 99% B
Flow Rate:0.6 mL/min
Injection Temperature:4 C
Internal Standard:See data dictionary
Retention Time:See data dictionary
Sample Injection:5.0 uL
Solvent A:40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Solvent B:90% isopropanol/10% acetonitrile; 10mM acetic acid; 10mM ammonium acetate
Analytical Time:13 min
Capillary Voltage:3500 eV
Time Program:15 min
Weak Wash Solvent Name:Isopropanol
Strong Wash Solvent Name:Isopropanol
Target Sample Temperature:Autosampler temp 4 C
Randomization Order:Excel
Chromatography Type:Reversed phase

MS:

MS ID:MS000896
Analysis ID:AN001001
Instrument Name:Agilent 6530 QTOF
Instrument Type:QTOF
MS Type:ESI
Ion Mode:POSITIVE
Capillary Voltage:3500 eV
Collision Energy:25 eV
Collision Gas:Nitrogen
Dry Gas Flow:8L/min
Dry Gas Temp:325 C
Fragment Voltage:120 eV
Fragmentation Method:Auto MS/MS
Ion Source Temperature:325 C
Ion Spray Voltage:1000
Ionization:Pos
Precursor Type:Intact Molecule
Reagent Gas:Nitrogen
Source Temperature:325 C
Dataformat:.d
Desolvation Gas Flow:11 L/min
Desolvation Temperature:350 C
Nebulizer:35 psig
Octpole Voltage:750
Resolution Setting:Exteded Dyamic Range
Scan Range Moverz:60-1700 Da
Scanning Cycle:2 Hz
Scanning Range:60-1700 Da
Skimmer Voltage:65
  
MS ID:MS000897
Analysis ID:AN001002
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
Ion Mode:NEGATIVE
Capillary Voltage:3500 eV
Collision Energy:25 eV
Collision Gas:Nitrogen
Dry Gas Flow:13L/min
Dry Gas Temp:200 C
Fragment Voltage:175 eV
Fragmentation Method:Auto MS/MS
Ion Source Temperature:325 C
Ion Spray Voltage:1000
Ionization:Neg
Precursor Type:Intact Molecule
Reagent Gas:Nitrogen
Source Temperature:325 C
Dataformat:.d
Desolvation Gas Flow:11 L/min
Desolvation Temperature:350 C
Nebulizer:35 psig
Octpole Voltage:750
Resolution Setting:Exteded Dyamic Range
Scan Range Moverz:60-1700 Da
Scanning Cycle:2 Hz
Scanning Range:60-1700 Da
Skimmer Voltage:65
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