Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA246676

Local Sample ID:	ile_gf_12_M_1
Subject ID:	SU002555
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	3-12 weeks
Animal Animal Supplier:	In house breeding
Animal Housing:	Germ-free and gnotobiotic isolators for germ and gnotobiotic mice. Individually ventilated cages for specific pathogen free mice
Animal Light Cycle:	12 hours
Animal Feed:	LabDiet (Cat# 5010; Laboratory Autoclavable Rodent Diet)

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Subject:

Subject ID:	SU002555
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	3-12 weeks
Animal Animal Supplier:	In house breeding
Animal Housing:	Germ-free and gnotobiotic isolators for germ and gnotobiotic mice. Individually ventilated cages for specific pathogen free mice
Animal Light Cycle:	12 hours
Animal Feed:	LabDiet (Cat# 5010; Laboratory Autoclavable Rodent Diet)

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
ile_gf_12_M_1	SA246676	FL030943	Ileum_content	Site
ile_gf_12_M_1	SA246676	FL030943	gf	Microbiota
ile_gf_12_M_1	SA246676	FL030943	12	Age(weeks)
ile_gf_12_M_1	SA246676	FL030943	M	Sex

Collection:

Collection ID:	CO002548
Collection Summary:	Mice were anaesthetized with isoflurane and blood was collected by retro-orbital bleed to serum separation collection tubes (BD, NJ, USA). Urine was collected from the mouse either before it was euthanized, immediately following euthanization or directly from the bladder following euthanization. Mice were euthanized by cervical dislocation. 1.5mL of ice-cold sterile PBS was injected into the peritoneal cavity, massaged for 20 seconds then removed and collected. The spleen and a segment of left lobe of the liver was collected. The intestine was excised and content from the entire length of the jejunum, ileum, cecum, and colon were collected. For the purposes of these experiments, the first 10cm of the small intestine was considered duodenum, and the remaining length was split in half with the proximal segment considered jejunum and the distal segment considered ileum. Following collection, blood was centrifuged (10,000 xg, 10 minutes, 4°C) and serum transferred into a 1.5mL Eppendorf tube. All collected samples were placed in liquid nitrogen immediately after collection and stored at -80°C until processing. For processing liver tissue, spleen, and intestinal content, ~50mg of tissue or content was added to a pre-weighed 2mL safe-lock tube containing a steel bead (3mm, Qiagen, Hilden, Germany). Tubes were reweighed and 5X v/w of ice-cold 50% methanol was added. Samples were homogenized (2 minutes, 30Hz), beads were removed, and samples stored at -20°C for 1 hour. Samples were centrifuged (max. speed, 15 minutes, 4֯C), supernatant was recovered and combined 1:4 with 50% methanol to obtain a final dilution of D20. For serum and urine, sample was combined 1:1 with 100% methanol and stored and -20°C for 1 hour. Samples were centrifuged (max. speed, 15 minutes, 4°C), supernatant was recovered and combined 1:25 with 50% methanol to obtain a final dilution of D50. Samples were centrifuged (max. speed, 15 minutes, 4°C) and supernatants were recovered and stored at -80°C until further processing.
Sample Type:	intestine content / liver / spleen / urine / peritoneal fluid / serum
Storage Conditions:	Described in summary

Treatment:

Treatment ID:	TR002567
Treatment Summary:	GF and OMM12-colonized gnotobiotic mice were housed in flexible-film isolators within the International Microbiome Centre (IMC) and SPF mice were maintained in the Mouse Barrier Unit (MBU) at the University of Calgary. GF mice were routinely tested for the absence of bacteria by aerobic and anaerobic culture, gram staining, and vital dye (DNA-dye Sytox green) staining of caecal contents and all mice were routinely screened for the presence of pathogens. For SPF colonization by co-housing, 8-week-old GF animals were exported, transferred into sterile individually ventilated cages and transported from the IMC to the MBU where a colonizer SPF mouse was transferred into each cage. For OMM12 colonization by co-housing, 8-week-old GF animals were transferred into isolators containing OMM12-colonized mice and an OMM12-colonized mouse was added to each cage. All animals were fed identical autoclaved diets and maintained with 12-hour light-dark cycle.

Treatment ID:

TR002567

Treatment Summary:

GF and OMM12-colonized gnotobiotic mice were housed in flexible-film isolators within the International Microbiome Centre (IMC) and SPF mice were maintained in the Mouse Barrier Unit (MBU) at the University of Calgary. GF mice were routinely tested for the absence of bacteria by aerobic and anaerobic culture, gram staining, and vital dye (DNA-dye Sytox green) staining of caecal contents and all mice were routinely screened for the presence of pathogens. For SPF colonization by co-housing, 8-week-old GF animals were exported, transferred into sterile individually ventilated cages and transported from the IMC to the MBU where a colonizer SPF mouse was transferred into each cage. For OMM12 colonization by co-housing, 8-week-old GF animals were transferred into isolators containing OMM12-colonized mice and an OMM12-colonized mouse was added to each cage. All animals were fed identical autoclaved diets and maintained with 12-hour light-dark cycle.

Sample Preparation:

Sampleprep ID:	SP002561
Sampleprep Summary:	Mice were anaesthetized with isoflurane and blood was collected by retro-orbital bleed to serum separation collection tubes (BD, NJ, USA). Urine was collected from the mouse either before it was euthanized, immediately following euthanization or directly from the bladder following euthanization. Mice were euthanized by cervical dislocation. 1.5mL of ice-cold sterile PBS was injected into the peritoneal cavity, massaged for 20 seconds then removed and collected. The spleen and a segment of left lobe of the liver was collected. The intestine was excised and content from the entire length of the jejunum, ileum, cecum, and colon were collected. For the purposes of these experiments, the first 10cm of the small intestine was considered duodenum, and the remaining length was split in half with the proximal segment considered jejunum and the distal segment considered ileum. Following collection, blood was centrifuged (10,000 xg, 10 minutes, 4°C) and serum transferred into a 1.5mL Eppendorf tube. All collected samples were placed in liquid nitrogen immediately after collection and stored at -80°C until processing.

Sampleprep ID:

SP002561

Sampleprep Summary:

Mice were anaesthetized with isoflurane and blood was collected by retro-orbital bleed to serum separation collection tubes (BD, NJ, USA). Urine was collected from the mouse either before it was euthanized, immediately following euthanization or directly from the bladder following euthanization. Mice were euthanized by cervical dislocation. 1.5mL of ice-cold sterile PBS was injected into the peritoneal cavity, massaged for 20 seconds then removed and collected. The spleen and a segment of left lobe of the liver was collected. The intestine was excised and content from the entire length of the jejunum, ileum, cecum, and colon were collected. For the purposes of these experiments, the first 10cm of the small intestine was considered duodenum, and the remaining length was split in half with the proximal segment considered jejunum and the distal segment considered ileum. Following collection, blood was centrifuged (10,000 xg, 10 minutes, 4°C) and serum transferred into a 1.5mL Eppendorf tube. All collected samples were placed in liquid nitrogen immediately after collection and stored at -80°C until processing.

Combined analysis:

Analysis ID	AN004021
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	Thermo Syncronis HILIC (100 x 2.1mm, 1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	NEGATIVE
Units	AUC

Chromatography:

Chromatography ID:	CH002971
Chromatography Summary:	All metabolomics data were collected at the Calgary Metabolomics Research Facility (CMRF). Briefly, samples were centrifuged (20,817 xg, 15 minutes, 4°C), then 200µL was transferred to a deep-well 96-well plate (Thermo FisherF) for LC-MS analysis. Data were collected on a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo-Fisher) coupled to a Vanquish™ UHPLC System (Thermo-Fisher). Metabolites were chromatographically separated on Syncronis HILIC UHPLC column (2.1mm x 100mm x 1.7um, Thermo-Fisher) at the flow rate of 600uL/min using a binary solvent system (solvent A, 20mM ammonium formate pH 3.0 in MS grade H20 and solvent B, MS grade acetonitrile with 0.1% formic acid (%v/v)) and the following gradient: 0-2 mins, 100 %B; 2-7 mins, 100-80 %B; 7-10 mins, 80-5 %B; 10-12 mins, 5% B; 12-13 mins, 5-100 %B; 13-15 mins, 100 %B. The samples injection volume was 2uL. MassM data were acquired in MS1, negative full scan mode at a resolution of 240,000 scanning from 50-750m/z.
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Syncronis HILIC (100 x 2.1mm, 1.7um)
Column Temperature:	30C
Flow Gradient:	2 stage linear gradient
Flow Rate:	600uL/min
Sample Injection:	2uL
Solvent A:	100% water; 20mM ammonium formate (pH3)
Solvent B:	100% acetonitrile; 0.1% formic acid
Analytical Time:	15 minutes
Washing Buffer:	10% MeOH in H2O + 0.1% formic acid
Target Sample Temperature:	4
Chromatography Type:	HILIC

MS:

MS ID:	MS003768
Analysis ID:	AN004021
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolomics data files were processed with ms-mint (version 0.1.8.3; mint.resistancedb.org) Python package for targeted metabolomics and verified using El-Maven software package. For targeted analysis, metabolites were identified by matching observed m/z signals and chromatographic retention times to those observed from commercial metabolite standards library (MSMLSTM Sigma-Aldrich) containing 639 standards, 397 of which were detectable using our LC-MS and sample preparation methods and 140 of which were observed a detectable levels in the biological samples.
Ion Mode:	NEGATIVE