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MB Sample ID: SA252928
Local Sample ID: | LI_El15644_6 |
Subject ID: | SU002612 |
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: | SU002612 |
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 ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
LI_El15644_6 | SA252928 | FL032281 | LI | SampleType |
LI_El15644_6 | SA252928 | FL032281 | 15644 | Group |
Collection:
Collection ID: | CO002605 |
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. Samples were collected of ileal, cecal, and colonic contents. |
Sample Type: | Intestine |
Collection Frequency: | single time point (72 hours) |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR002624 |
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: | SP002618 |
Sampleprep Summary: | Intestinal samples (colon, cecum, ileum) were prepared individually using a single protocol as follows. Samples were kept frozen on dry ice and massed to at least 10 mg. Four microliters of -20oC extraction solvent (2:2:1 methanol:acetonitrile:water + stable isotope labeled internal standards) were added per milligram of intestinal sample. Six to eight 1mm zirconia silica beads were added to each sample followed by prompt bead beating (15 Hz, for 10 minutes). Following a 1 hour incubation in the -20oC freezer, samples were centrifuged at 4oC at 18,407 rcf for 5 minutes. Supernatant was collected and stored at -20oC prior to centrifugal plate filtration (0.2 micron polyvinylidene difluoride (PVDF) Agilent Technologies, Santa Clara CA) at 4oC at 4,122 rcf for 3 min. Collection plate was sealed and maintained at 4oC prior to prompt analysis. 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 | AN004136 | AN004137 |
---|---|---|
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: | CH003064 |
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: | MS003883 |
Analysis ID: | AN004136 |
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: | MS003884 |
Analysis ID: | AN004137 |
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 |