Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA016197

Local Sample ID:	Blood436
Subject ID:	SU000384
Subject Type:	mouse
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57Bl/6
Age Or Age Range:	4.5 mo
Weight Or Weight Range:	29.1 g-49.2 g
Gender:	male
Animal Animal Supplier:	JAX
Animal Housing:	4/cage
Animal Feed:	Purina chow
Species Group:	Mammal

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Subject:

Subject ID:	SU000384
Subject Type:	mouse
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57Bl/6
Age Or Age Range:	4.5 mo
Weight Or Weight Range:	29.1 g-49.2 g
Gender:	male
Animal Animal Supplier:	JAX
Animal Housing:	4/cage
Animal Feed:	Purina chow
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Blood436	SA016197	FL003540	yes	HSV-1
Blood436	SA016197	FL003540	LF	Diet at Week 2

Collection:

Collection ID:	CO000378
Collection Summary:	For extraction, 500 µL of an ice-cold solution of 90:10 Methanol:Chloroform (v/v) ¬was added to the tubes containing the thawed cell pellets. MagNA lyser ceramic beads were added to the tubes, and a MagNA lyser was used to beat the samples for two 30 seconds pulses at 2,000 rpm placing samples on cold block for 5 min in between pulses. Sonication for 5 min was performed to the beaten cell samples followed by centrifugation at room temperature and at 16,000rcf for 4 min. A 350 µL aliquot of each experimental cell sample homogenized supernatant was transferred to a labeled 2.0 mL Lo-Bind Eppendorf tube.
Sample Type:	Mononuclear cells

Treatment:

Treatment ID:	TR000398
Treatment Summary:	3-week old mice were placed on a 10% low fat (LF) diet and acclimated for one week prior to intranasal HSV-1 infection or mock infection with PBS. Fourteen days post-infection mice were randomized to either a 45% high fat diet (HF) or remained on the LF diet. Eight weeks post-diet transition, mice were euthanized and brain tissue samples were collected and processed for metabolomics.

Sample Preparation:

Sampleprep ID:	SP000391
Sampleprep Summary:	For extraction, 500 µL of an ice-cold solution of 90:10 Methanol:Chloroform (v/v) ¬was added to the tubes containing the thawed cell pellets. MagNA lyser ceramic beads were added to the tubes, and a MagNA lyser was used to beat the samples for two 30 seconds pulses at 2,000 rpm placing samples on cold block for 5 min in between pulses. Sonication for 5 min was performed to the beaten cell samples followed by centrifugation at room temperature and at 16,000rcf for 4 min. A 350 µL aliquot of each experimental cell sample homogenized supernatant was transferred to a labeled 2.0 mL Lo-Bind Eppendorf tube. Analytical pooled samples were created by combining a 125 µL aliquot from each experimental sample into a labeled 20 mL glass vial. The QC pooled sample was vortexed for 30 sec and 350 µL aliquots were transferred to 5 labeled 2.0 mL Lo-Bind eppendorf tubes. Next, the supernatants of the study and QC pooled samples were dried overnight using a lyophilizer. The residue was reconstituted in 50 µL of reconstitution buffer (95:5 Water:Methanol, v/v) and mixed on an analog vortex mixer for 1 min with speed set at 10. Then, the samples were centrifuged at room temperature for 4 min at 16,000 rcf, and the supernatants were transferred to autosampler vials for data acquisition.

Sampleprep ID:

SP000391

Sampleprep Summary:

For extraction, 500 µL of an ice-cold solution of 90:10 Methanol:Chloroform (v/v) ¬was added to the tubes containing the thawed cell pellets. MagNA lyser ceramic beads were added to the tubes, and a MagNA lyser was used to beat the samples for two 30 seconds pulses at 2,000 rpm placing samples on cold block for 5 min in between pulses. Sonication for 5 min was performed to the beaten cell samples followed by centrifugation at room temperature and at 16,000rcf for 4 min. A 350 µL aliquot of each experimental cell sample homogenized supernatant was transferred to a labeled 2.0 mL Lo-Bind Eppendorf tube. Analytical pooled samples were created by combining a 125 µL aliquot from each experimental sample into a labeled 20 mL glass vial. The QC pooled sample was vortexed for 30 sec and 350 µL aliquots were transferred to 5 labeled 2.0 mL Lo-Bind eppendorf tubes. Next, the supernatants of the study and QC pooled samples were dried overnight using a lyophilizer. The residue was reconstituted in 50 µL of reconstitution buffer (95:5 Water:Methanol, v/v) and mixed on an analog vortex mixer for 1 min with speed set at 10. Then, the samples were centrifuged at room temperature for 4 min at 16,000 rcf, and the supernatants were transferred to autosampler vials for data acquisition.

Combined analysis:

Analysis ID	AN000595	AN000596
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Synapt G2 Si ESI-Q-TOF	Synapt G2 Si ESI-Q-TOF
Column	Waters ACQUITY UPLC HSS T3	Waters ACQUITY UPLC HSS T3
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Waters Synapt G2 Si QTOF	Waters Synapt G2 Si QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	intensity	intensity

Chromatography:

Chromatography ID:	CH000426
Chromatography Summary:	UPLC was performed on a Waters Acquity UPLC with an Acquity HSS T3 column (2.1x 100mm x 1.8 µm) at 50 °C using the reversed-phase separation. Water with 0.1% formic acid (mobile phase A) and methanol with 0.1% formic acid (mobile phase B) were used as mobile phases and the metabolites were chromatographically separated using a gradient separation:
Methods Filename:	RTI-RCMRC-RP
Instrument Name:	Synapt G2 Si ESI-Q-TOF
Column Name:	Waters ACQUITY UPLC HSS T3
Column Temperature:	50
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS000531
Analysis ID:	AN000595
Instrument Name:	Waters Synapt G2 Si QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS000532
Analysis ID:	AN000596
Instrument Name:	Waters Synapt G2 Si QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE