Summary of Study ST001093

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000731. The data can be accessed directly via it's Project DOI: 10.21228/M8638T This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001093
Study TitleImpact of genetic suppression (shRNA) of ALDH1A1 expression in human colon cancer cell line (COLO320)
Study TypeUntargeted metabolomics
Study SummaryUsing a multi-omics approach, we have investigated the impact of genetic suppression (shRNA) of ALDH1A1 expression on transcriptomics, proteomics and untargeted metabolomics analyses in a human colon cancer cell line (COLO320). The present study (i) generates an integrative omic profile of scramble shRNA vs. ALDH1A1 shRNA COLO320 cells, and (ii) identifies possible alterations in biological pathways caused by suppression of ALDH1A1 expression.
Institute
Yale University
Last NameVasiliou
First NameVasilis
Address60 College str
Emailgeorgia.charkoftaki@yale.edu; vasilis.vasiliou@yale.edu
Phone203.737.8094
Submit Date2018-11-12
Num Groups2
Raw Data File Type(s)raw(Waters)
Analysis Type DetailLC-MS
Release Date2019-09-23
Release Version1
Vasilis Vasiliou Vasilis Vasiliou
https://dx.doi.org/10.21228/M8638T
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000731
Project DOI:doi: 10.21228/M8638T
Project Title:Impact of genetic suppression (shRNA) of ALDH1A1 expression in human colon cancer cell line (COLO320)
Project Type:Untargeted metabolomics analyses
Project Summary:Using a multi-omics approach, we have investigated the impact of genetic suppression (shRNA) of ALDH1A1 expression on transcriptomics, proteomics and untargeted metabolomics analyses in a human colon cancer cell line (COLO320). The present study (i) generates an integrative omic profile of scramble shRNA vs. ALDH1A1 shRNA COLO320 cells, and (ii) identifies possible alterations in biological pathways caused by suppression of ALDH1A1 expression.
Institute:Yale School of Public Health
Department:Environmental Health Sciences
Last Name:Vasiliou
First Name:Vasilis
Address:60 College str, New Haven, CT, 06520, USA
Email:georgia.charkoftaki@yale.edu; vasilis.vasiliou@yale.edu
Phone:2037857028
Funding Source:NIH

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:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Group name
SA074508X01222018_Colo320_20V_Lipid_POS_4KO ALDH1A1
SA074509X01222018_Colo320_20V_Lipid_POS_3KO ALDH1A1
SA074510X01222018_Colo320_20V_Lipid_POS_5KO ALDH1A1
SA074511X01222018_Colo320_20V_Lipid_POS_7KO ALDH1A1
SA074512X01222018_Colo320_20V_Lipid_POS_9KO ALDH1A1
SA074513X01222018_Colo320_20V_Lipid_POS_2KO ALDH1A1
SA074514X01222018_Colo320_20V_Lipid_POS_8KO ALDH1A1
SA074515X01222018_Colo320_20V_Lipid_POS_QC_12QC
SA074516X01222018_Colo320_20V_Lipid_POS_QC_11QC
SA074517X01222018_Colo320_20V_Lipid_POS_QC_13QC
SA074518X01222018_Colo320_20V_Lipid_POS_QC_14QC
SA074519X01222018_Colo320_20V_Lipid_POS_QC_16QC
SA074520X01222018_Colo320_20V_Lipid_POS_QC_15QC
SA074521X01222018_Colo320_20V_Lipid_POS_QC_10QC
SA074522X01222018_Colo320_20V_Lipid_POS_QC_09QC
SA074523X01222018_Colo320_20V_Lipid_POS_QC_08QC
SA074524X01222018_Colo320_20V_Lipid_POS_QC_07QC
SA074525X01222018_Colo320_20V_Lipid_POS_15WT Colo320
SA074526X01222018_Colo320_20V_Lipid_POS_11WT Colo320
SA074527X01222018_Colo320_20V_Lipid_POS_14WT Colo320
SA074528X01222018_Colo320_20V_Lipid_POS_10WT Colo320
SA074529X01222018_Colo320_20V_Lipid_POS_12WT Colo320
SA074530X01222018_Colo320_20V_Lipid_POS_16WT Colo320
SA074531X01222018_Colo320_20V_Lipid_POS_13WT Colo320
Showing results 1 to 24 of 24

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
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
Instrument Type:QTOF
MS Type:ESI
Ion Mode:POSITIVE
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