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MB Sample ID: SA074512
| Local Sample ID: | X01222018_Colo320_20V_Lipid_POS_9 |
| Subject ID: | SU001137 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | ALDH1A1 |
| Cell Passage Number: | 5 |
| Cell Counts: | 4 million |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001137 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | ALDH1A1 |
| Cell Passage Number: | 5 |
| Cell Counts: | 4 million |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| X01222018_Colo320_20V_Lipid_POS_9 | SA074512 | FL011443 | KO ALDH1A1 | Group name |
Collection:
| Collection ID: | CO001131 |
| Collection Summary: | the cell culture medium was removed from the culture dishes, and the cells were rinsed three times with ice-cold DPBS. For quenching and metabolite extraction, 1 mL ice-cold methanol was added to the plate and the cells were immediately removed from the culture dish by scraping. The cell suspension was collected into 2 mL Protein Lobind Eppendorf tubes that were placed on ice. After cell counting (ViaStain AOPI Staining Solution,cridine orange/propidium iodide Nexcelom Bioscience, Lawrence, MA) using an automated cellometer (Cellometer K2, Nexcelom Bioscience, Lawrence, MA), the cell number was adjusted to 4x106 cells/tube. The cells were lysed by the application of a freeze-thaw cycle (freezing in liquid nitrogen for 5 min, thawing in warm water for 5 min), followed by sonication for 2 min. This process was repeated three times. The samples were then centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was transferred to 2 mL tubes and dried using a Speed Vac (SPD111V Savant, Thermo Fisher Scientific). The samples were then reconstituted in isopropanol:acetonitrile:water (2:1:1 %v/v) and 2.5 μL Splash® Lipidomix® (Avanti Polar Lipids Inc.) was added to each sample as an internal standard |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR001151 |
| Treatment Summary: | Cellular ALDH1A1 expression was suppressed using MISSION shRNA lentiviral transduction particles containing validated ALDH1A1 shRNA in a pLKO plasmid vector. Scramble control shRNA in pLKO plasmid vector was used to generate control cells (Sigma-Aldrich, St. Louis, MO). The shRNA transfection was carried out as described previously [1] and individual stable clones were selected using puromycin (10 μg/mL). Western blot analysis was used to verify changes in ALDH1A1 expression using methods described previously [2]. References [1] S. Guo, H. Bai, C.M. Megyola, S. Halene, D.S. Krause, D.T. Scadden, J. Lu, Complex oncogene dependence in microRNA-125a-induced myeloproliferative neoplasms, Proc Natl Acad Sci U S A, 109 (2012) 16636-16641. [2] Y. Chen, D.C. Thompson, V. Koppaka, J.V. Jester, V. Vasiliou, Ocular aldehyde dehydrogenases: protection against ultraviolet damage and maintenance of transparency for vision, Prog Retin Eye Res, 33 (2013) 28-39. |
Sample Preparation:
| Sampleprep ID: | SP001144 |
| Sampleprep Summary: | Briefly, the cell culture medium was removed from the culture dishes, and the cells were rinsed three times with ice-cold DPBS. For quenching and metabolite extraction, 1 mL ice-cold methanol was added to the plate and the cells were immediately removed from the culture dish by scraping. The cell suspension was collected into 2 mL Protein Lobind Eppendorf tubes that were placed on ice. After cell counting (ViaStain AOPI Staining Solution,cridine orange/propidium iodide Nexcelom Bioscience, Lawrence, MA) using an automated cellometer (Cellometer K2, Nexcelom Bioscience, Lawrence, MA), the cell number was adjusted to 4x106 cells/tube. The cells were lysed by the application of a freeze-thaw cycle (freezing in liquid nitrogen for 5 min, thawing in warm water for 5 min), followed by sonication for 2 min. This process was repeated three times. The samples were then centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was transferred to 2 mL tubes and dried using a Speed Vac (SPD111V Savant, Thermo Fisher Scientific). The samples were then reconstituted in isopropanol:acetonitrile:water (2:1:1 %v/v) and 2.5 μL Splash® Lipidomix® (Avanti Polar Lipids Inc.) was added to each sample as an internal standard. |
Combined analysis:
| Analysis ID | AN001779 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters |
| Column | Waters Acquity BEH C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Waters Synapt G2 XS QTOF |
| Ion Mode | POSITIVE |
| Units | abundance corresponding to m/z |
Chromatography:
| Chromatography ID: | CH001257 |
| Chromatography Summary: | The mobile phase involved a gradient comprising varying amounts of solutions A (10 mM ammonium formate in 60% v/v acetonitrile) and B (10 mM ammonium formate in 95% isopropanol and 5% acetonitrile). The linear gradient elution was: 40–43% B (0–2 min), 43–50% B (over 0.1 min), 50–54% B (over 10 min), 54–70% B (over 0.1 min), 70–99% B (over 5.9 min), 99-40% B (over 0.1 min), and 40% B (for 2 min), run at 300 μl/min in positive Electrospray Ionization mode and the column temperature was maintained at 55 °C |
| Instrument Name: | Waters |
| Column Name: | Waters Acquity BEH C18 (100 x 2.1mm,1.7um) |
| Flow Rate: | 300 μl/min |
| Injection Temperature: | 4 |
| Internal Standard: | SPLASH™ Lipidomix® Mass Spec Standard (Avanti Polar Lipids, Inc.) |
| Solvent A: | 60% acetonitrile/40% water; 10 mM ammonium formate |
| Solvent B: | 95% isopropanol/5% acetonitrile; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001643 |
| Analysis ID: | AN001779 |
| Instrument Name: | Waters Synapt G2 XS QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
