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MB Sample ID: SA052772
| Local Sample ID: | Control_2.3 |
| Subject ID: | SU000935 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | Daoy (ATCC® HTB-186™) |
| Subject Comments: | cancer cell line |
| Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000935 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | Daoy (ATCC® HTB-186™) |
| Subject Comments: | cancer cell line |
| Species Group: | Human |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Control_2.3 | SA052772 | FL010001 | Control | treatment |
Collection:
| Collection ID: | CO000929 |
| Collection Summary: | After 48 hours of transfection the cells were washed three times with ice-cold PBS (3X 5mL) and the cells were collected using a cell scraper. |
| Sample Type: | Cell |
| Collection Method: | Scraper |
| Tissue Cell Identification: | Daoy cell |
| Tissue Cell Quantity Taken: | protein normalization |
Treatment:
| Treatment ID: | TR000949 |
| Treatment Summary: | siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 according to the supplier’s protocol for 48 hours |
| Cell Growth Container: | plate |
| Cell Growth Config: | attached |
| Cell Growth Rate: | 36 hours |
| Cell Inoc Proc: | lipofectamine 3000 |
| Cell Media: | DMEM + GlutaMAX medium (Gibco), supplemented with 10% v/v foetal bovine serum (FBS, Gibco) and penicillin/streptomycin (1 U/ml, Gibco), |
| Cell Envir Cond: | 37°C in humidified 5% CO2 |
| Cell Harvesting: | After 48 h of transfection the cells were washed three times with ice-cold PBS (3x5 mL) and the celles were collected using a cell scraper. Ice-cold water (3 mL,sterile, MilliQ) was added to the samples. The suspension was transferred to a 15 mL polypropylene tube and then snap-frozen in liquid N2 and stored on ice for 5 min. The cells were lysed by two freeze-thaw cycles where the cells were thawed at 37°C in a water bath and frozen using liquid N2. Subsequently, the cells were sonicated in a |
| Cell Pct Confluence: | 80-90% |
| Cell Media Lastchanged: | 48 hours |
Sample Preparation:
| Sampleprep ID: | SP000942 |
| Sampleprep Summary: | The samples were thawed at room temperature and subjected to centrifugation for 17 min at 3000 rpm and 4°C. A quality control (QC) sample was created by pooling an equal volume from all samples. The aqueous supernatants were transferred to clean extraction tubes followed by addition of chloroform and methanol for the final proportion 2.85:4:4 water:methanol:chloroform. The extraction tubes were gently vortexed and then stored at 8°C for 20 min prior to centrifugation for 20 min at 3000 rpm and 4°C. The aqueous phases were recovered and evaporated to dryness at 40°C under N2. All samples were stored at -80°C after evaporation. Prior to analysis the samples were reconstituted in acetonitrile:Milli-Q water 90:10 |
| Processing Method: | lysis, freeze thaw cycles and sonication (see cell harvesting) |
| Processing Storage Conditions: | Room temperature and 8°C during extraction, -80°C before and after extraction |
| Extraction Method: | Liquid Liquid Extraction using CHCl3:MeOH:H2O 4:4:2.85 |
| Extract Storage: | Extracts were stored in -80°C after evaporation |
| Sample Resuspension: | Acetonitrile:Milli-Q water 90:10 |
| Cell Type: | Daoy cells |
Combined analysis:
| Analysis ID | AN001460 | AN001461 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Waters Acquity I-Class | Waters Acquity I-Class |
| Column | Waters Acquity BEH Amide (50 x 2.1mm,1.7um) | Waters Acquity BEH Amide (50 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Waters Synapt G2 QTOF | Waters Synapt G2 QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak Area | Peak Area |
Chromatography:
| Chromatography ID: | CH001027 |
| Chromatography Summary: | The samples were analyzed on a Acquity UPLC I-class system from Waters. A HILIC-Amide column (1.7 μm, i.d. 2.1x50 mm) from Waters was used for sample separation and a non-linear gradient elution profile from 100 % A to 100 % B was used. Mobile phase A consisted of 90:10 acetonitrile/water with 10 mM ammonium formate and 0.1% FA while mobile phase B consisted of 50:50 acetonitrile/ water with 10 mM ammonium formate and 0.1% FA. The flow rate was 0.3 ml/min, the column temperature 40°C and the injection volume 5 µL. |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | Waters Acquity BEH Amide (50 x 2.1mm,1.7um) |
| Column Temperature: | 40°C |
| Flow Gradient: | Non-linear gradient elution profile from 100 % A to 100 % B was used. In detail; 100% A was kept for 0.5 min then decreased non-linearly (slope-factor 8 in MassLynx) over 12.5 min to 100% B, 100% B was held for 3 min followed by 7 min at 100 % A to re-equilibrate the column for a total run-time of 23 min. |
| Flow Rate: | 0.3 ml/min |
| Injection Temperature: | 4°C |
| Sample Injection: | 5 μl |
| Solvent A: | 90% acetonitrile/10% water; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 50% acetonitrile/50% water; 0.1% formic acid; 10 mM ammonium formate |
| Analytical Time: | 23 min total sample run-time |
| Randomization Order: | All samples were analyzed in a randomized order with three QC injections interspaced every eleventh injection |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS001348 |
| Analysis ID: | AN001460 |
| Instrument Name: | Waters Synapt G2 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The raw data was converted to NetCDF files using the software DataBridge (Masslynx version 4.1, Waters). Peak detection and retention time alignment was performed using the R based software XCMS (Smith et al. 2006). The centWave function was used for peak detection and the function parameters were set as follows; the maximal deviation in m/z between scans was set to 8 ppm, the maximal and minimal peakwidth was set to 5 and 25 s respectively and the signal to noise ratio cutoff was set to 10. Retention time correction was performed using the “obiwarp” function. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 500°C |
| Capillary Voltage: | 1 kV |
| Collision Energy: | 20- 45 eV collision ramp, MSE acquistion |
| Source Temperature: | 120°C |
| Dataformat: | NetCDF |
| Scan Range Moverz: | m/z 50-800 |
| MS ID: | MS001349 |
| Analysis ID: | AN001461 |
| Instrument Name: | Waters Synapt G2 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The raw data was converted to NetCDF files using the software DataBridge (Masslynx version 4.1, Waters). Peak detection and retention time alignment was performed using the R based software XCMS (Smith et al. 2006). The centWave function was used for peak detection and the function parameters were set as follows; the maximal deviation in m/z between scans was set to 8 ppm, the maximal and minimal peakwidth was set to 5 and 25 s respectively and the signal to noise ratio cutoff was set to 10. Retention time correction was performed using the “obiwarp” function. |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 450°C |
| Capillary Voltage: | 2 kV |
| Collision Energy: | 20- 45 eV collision ramp, MSE acquistion |
| Source Temperature: | 120°C |
| Dataformat: | NetCDF |
| Scan Range Moverz: | m/z 50-800 |
