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MB Sample ID: SA252690
Local Sample ID: | PTJ0182_1 |
Subject ID: | SU002610 |
Subject Type: | Bacteria |
Subject Species: | Eggerthella lenta |
Taxonomy ID: | 84112 |
Genotype Strain: | various (see sample table) |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002610 |
Subject Type: | Bacteria |
Subject Species: | Eggerthella lenta |
Taxonomy ID: | 84112 |
Genotype Strain: | various (see sample table) |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
PTJ0182_1 | SA252690 | FL032171 | - | Strain |
PTJ0182_1 | SA252690 | FL032171 | - | Strain name |
PTJ0182_1 | SA252690 | FL032171 | - | Condition |
PTJ0182_1 | SA252690 | FL032171 | - | PlateNum |
Collection:
Collection ID: | CO002603 |
Collection Summary: | For the comparative strain metabolomics experiment, 96-well polypropylene deep well plates were prepared with 800μL of fresh media in each well. Starter cultures and inocula for 29 isolates of Eggerthella lenta and 1 isolate of Eggerthella sinensis [26] were prepared as described above for growth assays, except without final dilution, and 80 μL was used to inoculate wells, leaving a blank well in between every culture well to prevent cross-contamination. After 72 hours, OD600 measurements were taken, plates were centrifuged, and supernatants were collected. Plates were centrifuged at 1,928 rcf at 4°C for 8 minutes, after which supernatants were collected into fresh polypropylene tubes or plates, sealed, and flash-frozen in liquid nitrogen. |
Sample Type: | Bacterial culture supernatant |
Collection Frequency: | single time point (72 hours) |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR002622 |
Treatment Summary: | For growth and metabolomics experiments, glycerol stocks of the 3 E. lenta strains were first streaked on BHI+ agar plates and incubated at 37°C for 2-3 days. Individual colonies were inoculated into 3-4 mL liquid BHI+ and incubated at 37°C for 40-48 hours, or until approximately early stationary phase. Culture optical density (600 nm wavelength absorbance, OD600) was measured using a Hach DR1900 spectrophotometer. 1 mL samples of BHI starter cultures were then centrifuged at 1,568 rcf for 4 minutes in a microcentrifuge (ThermoScientific mySpin 12) in the anaerobic chamber and resuspended in 1 mL sterile phosphate-buffered saline (PBS). The resulting suspension was vortexed and diluted to an approximate OD600 of 0.1, and used as inoculum into defined experimental conditions. Varying media conditions were prepared separately and all allowed to fully reduce in the anaerobic chamber prior to inoculation. |
Sample Preparation:
Sampleprep ID: | SP002616 |
Sampleprep Summary: | Bacterial culture supernatant and sterile media, used in culture, were thawed on wet ice. Once thawed, samples were homogenized by inversion five times. Extracellular culture supernatant samples were prepared as follows: 20 μL of culture supernatant were extracted using 80 μL of a chilled extraction solvent at −20°C (1:1 acetonitrile:methanol, 5% water containing stable isotope-labeled internal standards). Samples were homogenized via pipette action, incubated for 1 hour at −20°C, centrifuged at 4°C at 6000 rcf for 5 min. The supernatant was transferred to a new plate and immediately sealed and kept at 4°C prior to prompt analysis via LC-MS/MS. |
Combined analysis:
Analysis ID | AN004133 | AN004134 |
---|---|---|
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: | CH003062 |
Chromatography Summary: | Samples, sterile media, 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: | MS003880 |
Analysis ID: | AN004133 |
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: | MS003881 |
Analysis ID: | AN004134 |
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 |