Return to study ST002747 main page
MB Sample ID: SA289280
Local Sample ID: | Inf5_infected_NA_5__014 |
Subject ID: | SU002854 |
Subject Type: | Cultured cells |
Subject Species: | Rickettsia parkeri; Homo sapiens |
Taxonomy ID: | 35792; 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002854 |
Subject Type: | Cultured cells |
Subject Species: | Rickettsia parkeri; Homo sapiens |
Taxonomy ID: | 35792; 9606 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Inf5_infected_NA_5__014 | SA289280 | FL035667 | Rickettsia parkeri & human | Genotype |
Inf5_infected_NA_5__014 | SA289280 | FL035667 | PostInfection | Treatment |
Collection:
Collection ID: | CO002847 |
Collection Summary: | A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018). |
Sample Type: | A549 cells |
Treatment:
Treatment ID: | TR002863 |
Treatment Summary: | A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018). |
Sample Preparation:
Sampleprep ID: | SP002860 |
Sampleprep Summary: | A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018). |
Combined analysis:
Analysis ID | AN004454 | AN004455 | AN004456 | AN004457 |
---|---|---|---|---|
Analysis type | MS | MS | MS | MS |
Chromatography type | HILIC | HILIC | Reversed phase | Reversed phase |
Chromatography system | Thermo Vanquish | Thermo Vanquish | 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) | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
MS Type | ESI | ESI | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
Units | counts, height | counts, height | counts, height | counts, height |
Chromatography:
Chromatography ID: | CH003345 |
Chromatography Summary: | HILIC |
Instrument Name: | Thermo Vanquish |
Column Name: | Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) |
Column Temperature: | 45C |
Flow Gradient: | Gradient elution was performed from 100% (B) at 0–2 min to 70% (B) at 7.7 min, 40% (B) at 9.5 min, 30% (B) at 10.25 min, 100% (B) at 12.75 min, isocratic until 16.75 min with a column flow of |
Flow Rate: | 0.4 mL/min. |
Solvent A: | 100% water; 10mM ammonium formate; 0.125% formic acid |
Solvent B: | 95% acetonitrile; 10mM ammonium formate; 0.125% formic acid |
Chromatography Type: | HILIC |
Chromatography ID: | CH003346 |
Chromatography Summary: | Lipids (ammonium acetate replaces ammonium formate in neg mode analysis |
Instrument Name: | Thermo Vanquish |
Column Name: | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
Column Temperature: | 65C |
Flow Gradient: | Gradient elution was performed from 15% (B) at 0 min to 30% (B) at 2 min, 48% (B) at 2.5 min, 82% (B) at 11 min, 99% (B) at 11.5 min, isocratic until 12 and resetting to initial conditions at 12.1 min through the end of the run at 15 min. |
Flow Rate: | is 0.6 mL/min. |
Solvent A: | 60% acetonitrile; 10 mM ammonium formate; 0.1% formic acid |
Solvent B: | 10% acetonitrile/90% isopropanol; 10 mM ammonium formate; 0.1% formic acid |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS004201 |
Analysis ID: | AN004454 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from the 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 1.0 Da. Collision energy was NCE 20, 50, 100 eV. |
Ion Mode: | POSITIVE |
MS ID: | MS004202 |
Analysis ID: | AN004455 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from the 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 1.0 Da. Collision energy was NCE 20, 50, 100 eV. |
Ion Mode: | NEGATIVE |
MS ID: | MS004203 |
Analysis ID: | AN004456 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected. Mass range was 220-1600 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 40, 60 eV. |
Ion Mode: | POSITIVE |
MS ID: | MS004204 |
Analysis ID: | AN004457 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected. Mass range was 220-1600 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 40, 60 eV. |
Ion Mode: | NEGATIVE |