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MB Sample ID: SA235213

Local Sample ID:H3
Subject ID:SU002431
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57Bl/6
Age Or Age Range:20 weeks (at euthanasia)
Gender:Male and female
Animal Animal Supplier:Jackson Labratories
Animal Light Cycle:Standard light dark cycle
Animal Feed:PicoLab Rodent Diet 20, 20% protein

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Subject:

Subject ID:SU002431
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57Bl/6
Age Or Age Range:20 weeks (at euthanasia)
Gender:Male and female
Animal Animal Supplier:Jackson Labratories
Animal Light Cycle:Standard light dark cycle
Animal Feed:PicoLab Rodent Diet 20, 20% protein

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
H3SA235213FL029180FemaleSex
H3SA235213FL029180Germ-FreeMicrobiome Status

Collection:

Collection ID:CO002424
Collection Summary:Samples were collected following mouse euthanasia. The humerus was obtained and bone marrow was flushed using PBS. Next, metabolites were extracted using methanol:acetone.
Sample Type:Bone

Treatment:

Treatment ID:TR002443
Treatment Summary:All C57Bl/6J mice (male, female, germ-free, conventional) were housed at Montana State University. During this time, mice were kept on a light/dark cycle and fed a standard chow diet ad libitum. Upon 20 weeks of age, mice were euthanized. No additional factors such as injury, diet, or exercise occurred. Mice were employed to analyze differences in bone at macro and micro levels that are associated with the microbiome and sex.

Sample Preparation:

Sampleprep ID:SP002437
Sampleprep Summary:Once euthanized, tissues were dissected and harvested for various techniques. For metabolomics, humeri samples underwent homogenization/crushing, extracted with methanol:acetone, and were then subjected to rounds of vortexing and centrifugation. Once metabolites were isolated, samples were dried via vacuum concentration and resuspended with acetonitrile:water for LC-MS analysis.

Combined analysis:

Analysis ID AN003826
Analysis type MS
Chromatography type HILIC
Chromatography system Agilent 1290
Column Cogent Diamond Hydride (150 × 2.1mm,4um)
MS Type ESI
MS instrument type Single quadrupole
MS instrument name Agilent 6538 QTOF
Ion Mode POSITIVE
Units Peak Intensity

Chromatography:

Chromatography ID:CH002831
Chromatography Summary:Samples were analyzed using an Agilent 1290 UPLC coupled to an Agilent 6538 QTOF. All samples were run in normal phase using a Cogent Diamond Hydride HILIC column (150 x 2.1 mm; 100Å pore size on a 4µm particle size). Solvents: 0.1% formic acid in water (A), 0.1% formic acid in acetonitrile (B). Elution gradient: 100 to 25% solvent B over 17 minutes, 0 to 70% solvent A over 20 minutes, at 21 minutes, B increases from 25% to 100% till 25 minutes while A decreases from 70% to 0% till 25 minutes. The last 4 minutes act as a wash. Total run time = 25 minute method (per sample)
Instrument Name:Agilent 1290
Column Name:Cogent Diamond Hydride (150 × 2.1mm,4um)
Flow Gradient:100 to 25% solvent B over 17 minutes, 0 to 70% solvent A over 20 minutes, at 21 minutes, B increases from 25% to 100% till 25 minutes while A decreases from 70% to 0% till 25 minutes. The last 4 minutes act as a wash.
Flow Rate:0.400 mL/min
Injection Temperature:4C
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Analytical Time:25 minutes
Chromatography Type:HILIC

MS:

MS ID:MS003568
Analysis ID:AN003826
Instrument Name:Agilent 6538 QTOF
Instrument Type:Single quadrupole
MS Type:ESI
MS Comments:Data was acquired in positive mode following a 25-minute method. Following injection and acquisition, .d files were converted to .mzMLs using MSConvert (Threshold peak filter value = 1000; Peak picking = true, 1-1; 32-bit, no z-lib compression). Once .mzMLs were avaiable, XCMS was used (parameters reflected run parameters = positive mode, feature detection 15 ppm, adducts = [M+H]+, [M+Na]+
Ion Mode:POSITIVE
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