Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA063417

Local Sample ID:	H3
Subject ID:	SU001046
Subject Type:	Other
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Microbiome

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Subject:

Subject ID:	SU001046
Subject Type:	Other
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Microbiome

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
H3	SA063417	FL010854	High-Dose Tempol	Treatment

Collection:

Collection ID:	CO001040
Collection Summary:	6-week-old wild-type male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) were transferred into anaerobic chamber (Coy Laboratory Products, Inc., Grass Lake, MI) following CO2 asphyxiation. All the following procedures were performed under strict anaerobic conditions with an oxygen level below 20 ppm and cecal contents were collected
Sample Type:	Cecum

Treatment:

Treatment ID:	TR001060
Treatment Summary:	The cecal content suspension was treated with tempol at a final concentration 0.01 mg/mL, 0.1 mg/mL and 1 mg/mL, following a brief centrifugation and incubation at 37 °C for 4 h in dark.

Sample Preparation:

Sampleprep ID:	SP001053
Sampleprep Summary:	The microbiota suspension saved after 4h incubation was used for 1H NMR spectroscopy. 1 mL of microbiota suspension was centrifuged at low speed (700 g, 4 °C for 1 min) to pellet down large particles. The maximum supernatant volume was transferred to a new tube, centrifuged at high speed (6000 g, 4 °C for 3 min) to pellet down bacteria. The microbial pellet was washed two times with PBS. After the third wash, 1 mL of pre-cooled methanol:H2O (v/v = 2:1) and 1.0 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the microbial pellet, followed by homogenization (6500 rpm, 1 cycle, 60 s) using the Precellys tissue homogenizer (Bertin Technologies, Rockville, MD). The homogenized sample was freeze-thawed three times with liquid nitrogen and 37°C water bath, then was homogenized again and sonicated for 15 min at 250W with Branson 1510 Ultrasonic Cleaner (Branson Ultrasonics, Danbury, CT) to rupture microbial cell walls and release intracellular metabolites. The sample was centrifuged (11180g, 4 °C, and 10 min) and the supernatants was transferred to a new 2 mL tube. Another 1 mL methanol:H2O (v/v = 2:1) was added to the pellets and the extraction procedure was repeated. All supernatants were combined, dried down and reconstituted in 600 μL of PBS (K2HPO4/NaH2PO4, 0.1M, pH 7.4, containing 50% D2O and 0.005% TSP-d4 as internal standard). Following centrifugation (13 000g, 4 °C, 10min), 550 μL of each extract was transferred into a 5 mm NMR tube for analysis.

Sampleprep ID:

SP001053

Sampleprep Summary:

The microbiota suspension saved after 4h incubation was used for 1H NMR spectroscopy. 1 mL of microbiota suspension was centrifuged at low speed (700 g, 4 °C for 1 min) to pellet down large particles. The maximum supernatant volume was transferred to a new tube, centrifuged at high speed (6000 g, 4 °C for 3 min) to pellet down bacteria. The microbial pellet was washed two times with PBS. After the third wash, 1 mL of pre-cooled methanol:H2O (v/v = 2:1) and 1.0 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the microbial pellet, followed by homogenization (6500 rpm, 1 cycle, 60 s) using the Precellys tissue homogenizer (Bertin Technologies, Rockville, MD). The homogenized sample was freeze-thawed three times with liquid nitrogen and 37°C water bath, then was homogenized again and sonicated for 15 min at 250W with Branson 1510 Ultrasonic Cleaner (Branson Ultrasonics, Danbury, CT) to rupture microbial cell walls and release intracellular metabolites. The sample was centrifuged (11180g, 4 °C, and 10 min) and the supernatants was transferred to a new 2 mL tube. Another 1 mL methanol:H2O (v/v = 2:1) was added to the pellets and the extraction procedure was repeated. All supernatants were combined, dried down and reconstituted in 600 μL of PBS (K2HPO4/NaH2PO4, 0.1M, pH 7.4, containing 50% D2O and 0.005% TSP-d4 as internal standard). Following centrifugation (13 000g, 4 °C, 10min), 550 μL of each extract was transferred into a 5 mm NMR tube for analysis.

Analysis:

MB Sample ID:	SA063417
Analysis ID:	AN001649
Analysis Type:	NMR
Num Factors:	4
Num Metabolites:	14
Units:	ppm

NMR:

NMR ID:	NM000124
Analysis ID:	AN001649
Instrument Name:	Bruker
Instrument Type:	FT-NMR
NMR Experiment Type:	1D 1H
Spectrometer Frequency:	600 MHz