{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001093","ANALYSIS_ID":"AN001779","VERSION":"1","CREATED_ON":"November 13, 2018, 1:12 pm"},

"PROJECT":{"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"},

"STUDY":{"STUDY_TITLE":"Impact of genetic suppression (shRNA) of ALDH1A1 expression in human colon cancer cell line (COLO320)","STUDY_TYPE":"Untargeted metabolomics","STUDY_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","LAST_NAME":"Vasiliou","FIRST_NAME":"Vasilis","ADDRESS":"60 College str","EMAIL":"georgia.charkoftaki@yale.edu; vasilis.vasiliou@yale.edu","PHONE":"203.737.8094","NUM_GROUPS":"2"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","GENOTYPE_STRAIN":"ALDH1A1","CELL_PASSAGE_NUMBER":"5","CELL_COUNTS":"4 million"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_10",
"Factors":{"Group name":"WT Colo320"},
"Additional sample data":{"Label":"1"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_11",
"Factors":{"Group name":"WT Colo320"},
"Additional sample data":{"Label":"1"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_12",
"Factors":{"Group name":"WT Colo320"},
"Additional sample data":{"Label":"1"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_13",
"Factors":{"Group name":"WT Colo320"},
"Additional sample data":{"Label":"1"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_14",
"Factors":{"Group name":"WT Colo320"},
"Additional sample data":{"Label":"1"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_15",
"Factors":{"Group name":"WT Colo320"},
"Additional sample data":{"Label":"1"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_16",
"Factors":{"Group name":"WT Colo320"},
"Additional sample data":{"Label":"1"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_2",
"Factors":{"Group name":"KO ALDH1A1"},
"Additional sample data":{"Label":"2"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_3",
"Factors":{"Group name":"KO ALDH1A1"},
"Additional sample data":{"Label":"2"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_4",
"Factors":{"Group name":"KO ALDH1A1"},
"Additional sample data":{"Label":"2"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_5",
"Factors":{"Group name":"KO ALDH1A1"},
"Additional sample data":{"Label":"2"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_7",
"Factors":{"Group name":"KO ALDH1A1"},
"Additional sample data":{"Label":"2"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_8",
"Factors":{"Group name":"KO ALDH1A1"},
"Additional sample data":{"Label":"2"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_9",
"Factors":{"Group name":"KO ALDH1A1"},
"Additional sample data":{"Label":"2"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_07",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_08",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_09",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_10",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_11",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_12",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_13",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_14",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_15",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
},
{
"Subject ID":"-",
"Sample ID":"X01222018_Colo320_20V_Lipid_POS_QC_16",
"Factors":{"Group name":"QC"},
"Additional sample data":{"Label":"3"}
}
],
"COLLECTION":{"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_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."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"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","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters","COLUMN_NAME":"Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)","FLOW_RATE":"300 μl/min","SOLVENT_A":"10 mM ammonium formate in 60% v/v acetonitrile","SOLVENT_B":"10 mM ammonium formate in 95% isopropanol and 5% acetonitrile","INJECTION_TEMPERATURE":"4","INTERNAL_STANDARD":"SPLASH™ Lipidomix® Mass Spec Standard (Avanti Polar Lipids, Inc.)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Waters Synapt G2 XS QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_RESULTS_FILE":"ST001093_AN001779_Results.txt UNITS:abundance corresponding to m/z Has m/z:Yes Has RT:Yes RT units:Seconds"}

}