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MB Sample ID: SA252992

Local Sample ID:Serum_GF_2
Subject ID:SU002613
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:6-10 weeks
Gender:Male
Animal Animal Supplier:University of California, San Francisco Gnotobiotics core facility

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Subject:

Subject ID:SU002613
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:6-10 weeks
Gender:Male
Animal Animal Supplier:University of California, San Francisco Gnotobiotics core facility

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Serum_GF_2SA252992FL032293SerumSampleType
Serum_GF_2SA252992FL032293GFGroup

Collection:

Collection ID:CO002606
Collection Summary:Sample collection was performed as described in doi.org/10.1016/j.chom.2021.11.001 . Briefly, C57BL/6J mice (males, ages 4-8 weeks) were obtained from the University of California, San Francisco Gnotobiotics core facility (gnotobiotics.ucsf.edu) and housed in Iso positive cages (Tecniplast). Mice were colonized via oral gavage with E. lenta monocultures (109 CFU/mL, 200 μl gavage, n=6 E. lenta DSM 2243, n=5 E. lenta DSM 15644, n=4 E. lenta AB12n2) or control media (n=5 GF). Colonization was confirmed via anaerobic culturing and/or qPCR for an E. lenta specific marker (elnmrk1) [10,26]. Mice were colonized for 2 weeks prior to sacrifice and sample collection. Serum samples were collected.
Sample Type:Blood (serum)
Collection Frequency:single time point (2 weeks after colonization)
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002625
Treatment Summary:Mice were colonized via oral gavage with E. lenta monocultures (109 CFU/mL, 200 μl gavage, n=6 E. lenta DSM 2243, n=5 E. lenta DSM 15644, n=4 E. lenta AB12n2) or control media (n=5 GF). Colonization was confirmed via anaerobic culturing and/or qPCR for an E. lenta specific marker (elnmrk1). Mice were colonized for 2 weeks prior to sacrifice and sample collection. Samples were collected of ileal, cecal, and colonic contents, as well as serum.

Sample Preparation:

Sampleprep ID:SP002619
Sampleprep Summary:Serum samples were first thawed on wet ice. 20 μL of serum was extracted with 4 volumes of methanol, containing stable isotope labeled internal standards. Samples were homogenized by vortexing for 20 seconds and placed in a -20oC for 1 hour to maximize protein precipitation. After freezer incubation, samples were centrifuged at 4oC at 18,407 rcf for 5 minutes. Supernatant was removed and dried under vacuum via centrivap (Labconco Corp.). Dried samples were then resuspended in 30 μL of 80% acetonitrile in water containing exogenous standard CUDA at 60 ng/mL. Samples were maintained at 4oC prior to prompt analysis.

Combined analysis:

Analysis ID AN004138 AN004139
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Vanquish Thermo Vanquish
Column Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE NEGATIVE
Units relative ion counts relative ion counts

Chromatography:

Chromatography ID:CH003065
Chromatography Summary:Samples, pools, and blanks were promptly added to a Thermo Vanquish Autosampler at 4°C in a Vanquish UHPLC (Thermo Fisher Scientific, Waltham, MA). Chromatographic separation was performed using an ACQUITY Bridged Ethylene Hybrid (BEH) Amide column 2.1 x 150 mm, 1.7-micron particle size, (Waters Corp. Milford, MA), using chromatographic conditions published elsewhere (HILIC method described in the Supplementary Methods of doi.org/10.1038/s41586-021-03707-9).
Instrument Name:Thermo Vanquish
Column Name:Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:40
Flow Gradient:The gradient profile was held at 100% B for 2 minutes, from 100% B to 70% B in 5 minutes, holding at 70% B for 0.7 minute, from 70% B to 40% B for 1.3 minutes, holding at 40% B for 0.5 minutes, from 40% B to 30% B for 0.75 minutes, before returning to 100% B for 2.5 minutes and holding at 100% B for 4 minutes.
Flow Rate:400 μL per minute
Solvent A:100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3
Solvent B:95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate
Chromatography Type:HILIC

MS:

MS ID:MS003885
Analysis ID:AN004138
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. Metabolomics data was processed using MS-DIAL v4.60. Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average.
Ion Mode:POSITIVE
  
MS ID:MS003886
Analysis ID:AN004139
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. Metabolomics data was processed using MS-DIAL v4.60. Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average.
Ion Mode:NEGATIVE
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