Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA254551

Local Sample ID:	DVC T1DM 2
Subject ID:	SU002625
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002625
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
DVC T1DM 2	SA254551	FL032615	STZ	Treatment
DVC T1DM 2	SA254551	FL032615	dorsal vagal complex	Tissue Region

Collection:

Collection ID:	CO002618
Collection Summary:	Mice were euthanized by microwave fixation system at 5kW for 0.6 seconds (MMW-05, Muromachi Kikai Company, Japan). Brain regions (HIPP, CTX, DVC), as well as muscle and liver, were dissected postmortem.
Sample Type:	Brain

Treatment:

Treatment ID:	TR002637
Treatment Summary:	We designed a two-arm study, where euthanasia occurs through either 1) direct decapitation or 2) focused microwave (Fig. 1). In both arms, we performed rapid dissection of the brain followed by cryopreservation in liquid nitrogen. The major difference between the two arms is the tissue fixation or enzyme inactivation step occurs either during cryopreservation for decapitation (CP, ~90 seconds) or focused microwave (FM, ~0.6 second).

Treatment ID:

TR002637

Treatment Summary:

We designed a two-arm study, where euthanasia occurs through either 1) direct decapitation or 2) focused microwave (Fig. 1). In both arms, we performed rapid dissection of the brain followed by cryopreservation in liquid nitrogen. The major difference between the two arms is the tissue fixation or enzyme inactivation step occurs either during cryopreservation for decapitation (CP, ~90 seconds) or focused microwave (FM, ~0.6 second).

Sample Preparation:

Sampleprep ID:	SP002631
Sampleprep Summary:	Brains were removed immediately post-mortem, and washed once with PBS, twice with diH2O, blotted dry, and snap frozen in liquid nitrogen. Other set of brains were snap frozen after microwave fixation as described above. The frozen tissues were pulverized to 10 μm particles in liquid N2 using a Freezer/Mill Cryogenic Grinder (SPEX SamplePrep). Brain regions were extracted with 1ml of 50% methanol in the grinder, while for muscle and liver twenty milligrams of each pulverized tissue were extracted in 1ml of 50% methanol and separated into polar (aqueous layer), and protein/DNA/RNA/glycogen pellet. The polar fraction was dried at 10-3 mBar using a SpeedVac (Thermo) followed by derivatization.

Sampleprep ID:

SP002631

Sampleprep Summary:

Brains were removed immediately post-mortem, and washed once with PBS, twice with diH2O, blotted dry, and snap frozen in liquid nitrogen. Other set of brains were snap frozen after microwave fixation as described above. The frozen tissues were pulverized to 10 μm particles in liquid N2 using a Freezer/Mill Cryogenic Grinder (SPEX SamplePrep). Brain regions were extracted with 1ml of 50% methanol in the grinder, while for muscle and liver twenty milligrams of each pulverized tissue were extracted in 1ml of 50% methanol and separated into polar (aqueous layer), and protein/DNA/RNA/glycogen pellet. The polar fraction was dried at 10-3 mBar using a SpeedVac (Thermo) followed by derivatization.

Combined analysis:

Analysis ID	AN004159
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890A
Column	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5975C
Ion Mode	POSITIVE
Units	Relative abundance

Chromatography:

Chromatography ID:	CH003078
Instrument Name:	Agilent 7890A
Column Name:	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
Column Temperature:	60-325
Flow Gradient:	na
Flow Rate:	0.69 ml/min
Solvent A:	na
Solvent B:	na
Chromatography Type:	GC

MS:

MS ID:	MS003906
Analysis ID:	AN004159
Instrument Name:	Agilent 5975C
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	GCMS protocols were similar to those described previously with a modified temperature gradient was used for GC: Initial temperature was 130degC, held for 4 minutes, rising at 6degC/minutes to 243degC, rising at 60degC/minutes to 280degC, held for 2 minutes. The electron ionization (EI) energy was set to 70 eV. Scan (m/z:50-800) and full scan mode were used for metabolomics analysis. Mass spectra were translated to relative metabolite abundance using the Automated Mass Spectral Deconvolution and Identification System (AMDIS) software matched to the FiehnLib metabolomics library (available through Agilent) for retention time and fragmentation pattern matching with a confidence score of > 80 (Fiehn, 2016; Fiehn et al., 2000; Kind et al., 2009). Data was further analyzed using the Data Extraction for Stable Isotope-labelled Metabolites (DEXSI) software package. Untargeted metabolomics data was normalized to total ion chromatogram. For glucose tracer raw data was exported and correction for natural abundance was done by IsoCorrectoR. Fractional enrichment of each metabolite was calculated as the relative abundance of each isotopologue relative to the sum of all other isotopologues. Mean enrichment was calculated as sum of fractional enrichment of labeled isotopologues (M1, M2, M3…). For principal component analysis, pathway impact analysis the online tool Metaboanlyst was used (https://www.metaboanalyst.ca/). Data was auto scaled and log transformed.
Ion Mode:	POSITIVE