#METABOLOMICS WORKBENCH gcharkoftaki_20181112_062002 DATATRACK_ID:1568 STUDY_ID:ST001093 ANALYSIS_ID:AN001779 PROJECT_ID:PR000731 VERSION 1 CREATED_ON November 13, 2018, 1:12 pm #PROJECT PR:PROJECT_TITLE Impact of genetic suppression (shRNA) of ALDH1A1 expression in human colon PR:PROJECT_TITLE cancer cell line (COLO320) PR:PROJECT_TYPE Untargeted metabolomics analyses PR:PROJECT_SUMMARY Using a multi-omics approach, we have investigated the impact of genetic PR:PROJECT_SUMMARY suppression (shRNA) of ALDH1A1 expression on transcriptomics, proteomics and PR:PROJECT_SUMMARY untargeted metabolomics analyses in a human colon cancer cell line (COLO320). PR:PROJECT_SUMMARY The present study (i) generates an integrative omic profile of scramble shRNA PR:PROJECT_SUMMARY vs. ALDH1A1 shRNA COLO320 cells, and (ii) identifies possible alterations in PR:PROJECT_SUMMARY biological pathways caused by suppression of ALDH1A1 expression. PR:INSTITUTE Yale School of Public Health PR:DEPARTMENT Environmental Health Sciences PR:LAST_NAME Vasiliou PR:FIRST_NAME Vasilis PR:ADDRESS 60 College str, New Haven, CT, 06520, USA PR:EMAIL georgia.charkoftaki@yale.edu; vasilis.vasiliou@yale.edu PR:PHONE 2037857028 PR:FUNDING_SOURCE NIH #STUDY ST:STUDY_TITLE Impact of genetic suppression (shRNA) of ALDH1A1 expression in human colon ST:STUDY_TITLE cancer cell line (COLO320) ST:STUDY_TYPE Untargeted metabolomics ST:STUDY_SUMMARY Using a multi-omics approach, we have investigated the impact of genetic ST:STUDY_SUMMARY suppression (shRNA) of ALDH1A1 expression on transcriptomics, proteomics and ST:STUDY_SUMMARY untargeted metabolomics analyses in a human colon cancer cell line (COLO320). ST:STUDY_SUMMARY The present study (i) generates an integrative omic profile of scramble shRNA ST:STUDY_SUMMARY vs. ALDH1A1 shRNA COLO320 cells, and (ii) identifies possible alterations in ST:STUDY_SUMMARY biological pathways caused by suppression of ALDH1A1 expression. ST:INSTITUTE Yale ST:LAST_NAME Vasiliou ST:FIRST_NAME Vasilis ST:ADDRESS 60 College str ST:EMAIL georgia.charkoftaki@yale.edu; vasilis.vasiliou@yale.edu ST:PHONE 203.737.8094 ST:NUM_GROUPS 2 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN ALDH1A1 SU:CELL_PASSAGE_NUMBER 5 SU:CELL_COUNTS 4 million #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_10 Group name:WT Colo320 Label=1 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_11 Group name:WT Colo320 Label=1 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_12 Group name:WT Colo320 Label=1 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_13 Group name:WT Colo320 Label=1 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_14 Group name:WT Colo320 Label=1 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_15 Group name:WT Colo320 Label=1 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_16 Group name:WT Colo320 Label=1 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_2 Group name:KO ALDH1A1 Label=2 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_3 Group name:KO ALDH1A1 Label=2 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_4 Group name:KO ALDH1A1 Label=2 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_5 Group name:KO ALDH1A1 Label=2 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_7 Group name:KO ALDH1A1 Label=2 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_8 Group name:KO ALDH1A1 Label=2 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_9 Group name:KO ALDH1A1 Label=2 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_07 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_08 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_09 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_10 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_11 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_12 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_13 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_14 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_15 Group name:QC Label=3 SUBJECT_SAMPLE_FACTORS - X01222018_Colo320_20V_Lipid_POS_QC_16 Group name:QC Label=3 #COLLECTION CO:COLLECTION_SUMMARY the cell culture medium was removed from the culture dishes, and the cells were CO:COLLECTION_SUMMARY rinsed three times with ice-cold DPBS. For quenching and metabolite extraction, CO:COLLECTION_SUMMARY 1 mL ice-cold methanol was added to the plate and the cells were immediately CO:COLLECTION_SUMMARY removed from the culture dish by scraping. The cell suspension was collected CO:COLLECTION_SUMMARY into 2 mL Protein Lobind Eppendorf tubes that were placed on ice. After cell CO:COLLECTION_SUMMARY counting (ViaStain AOPI Staining Solution,cridine orange/propidium iodide CO:COLLECTION_SUMMARY Nexcelom Bioscience, Lawrence, MA) using an automated cellometer (Cellometer K2, CO:COLLECTION_SUMMARY Nexcelom Bioscience, Lawrence, MA), the cell number was adjusted to 4x106 CO:COLLECTION_SUMMARY cells/tube. The cells were lysed by the application of a freeze-thaw cycle CO:COLLECTION_SUMMARY (freezing in liquid nitrogen for 5 min, thawing in warm water for 5 min), CO:COLLECTION_SUMMARY followed by sonication for 2 min. This process was repeated three times. The CO:COLLECTION_SUMMARY samples were then centrifuged at 15,000 rpm for 10 min at 4°C, and the CO:COLLECTION_SUMMARY supernatant was transferred to 2 mL tubes and dried using a Speed Vac (SPD111V CO:COLLECTION_SUMMARY Savant, Thermo Fisher Scientific). The samples were then reconstituted in CO:COLLECTION_SUMMARY isopropanol:acetonitrile:water (2:1:1 %v/v) and 2.5 μL Splash® Lipidomix® CO:COLLECTION_SUMMARY (Avanti Polar Lipids Inc.) was added to each sample as an internal standard CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Cellular ALDH1A1 expression was suppressed using MISSION shRNA lentiviral TR:TREATMENT_SUMMARY transduction particles containing validated ALDH1A1 shRNA in a pLKO plasmid TR:TREATMENT_SUMMARY vector. Scramble control shRNA in pLKO plasmid vector was used to generate TR:TREATMENT_SUMMARY control cells (Sigma-Aldrich, St. Louis, MO). The shRNA transfection was carried TR:TREATMENT_SUMMARY out as described previously [1] and individual stable clones were selected using TR:TREATMENT_SUMMARY puromycin (10 μg/mL). Western blot analysis was used to verify changes in TR:TREATMENT_SUMMARY ALDH1A1 expression using methods described previously [2]. References [1] S. TR:TREATMENT_SUMMARY Guo, H. Bai, C.M. Megyola, S. Halene, D.S. Krause, D.T. Scadden, J. Lu, Complex TR:TREATMENT_SUMMARY oncogene dependence in microRNA-125a-induced myeloproliferative neoplasms, Proc TR:TREATMENT_SUMMARY Natl Acad Sci U S A, 109 (2012) 16636-16641. [2] Y. Chen, D.C. Thompson, V. TR:TREATMENT_SUMMARY Koppaka, J.V. Jester, V. Vasiliou, Ocular aldehyde dehydrogenases: protection TR:TREATMENT_SUMMARY against ultraviolet damage and maintenance of transparency for vision, Prog TR:TREATMENT_SUMMARY Retin Eye Res, 33 (2013) 28-39. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, the cell culture medium was removed from the culture dishes, and the SP:SAMPLEPREP_SUMMARY cells were rinsed three times with ice-cold DPBS. For quenching and metabolite SP:SAMPLEPREP_SUMMARY extraction, 1 mL ice-cold methanol was added to the plate and the cells were SP:SAMPLEPREP_SUMMARY immediately removed from the culture dish by scraping. The cell suspension was SP:SAMPLEPREP_SUMMARY collected into 2 mL Protein Lobind Eppendorf tubes that were placed on ice. SP:SAMPLEPREP_SUMMARY After cell counting (ViaStain AOPI Staining Solution,cridine orange/propidium SP:SAMPLEPREP_SUMMARY iodide Nexcelom Bioscience, Lawrence, MA) using an automated cellometer SP:SAMPLEPREP_SUMMARY (Cellometer K2, Nexcelom Bioscience, Lawrence, MA), the cell number was adjusted SP:SAMPLEPREP_SUMMARY to 4x106 cells/tube. The cells were lysed by the application of a freeze-thaw SP:SAMPLEPREP_SUMMARY cycle (freezing in liquid nitrogen for 5 min, thawing in warm water for 5 min), SP:SAMPLEPREP_SUMMARY followed by sonication for 2 min. This process was repeated three times. The SP:SAMPLEPREP_SUMMARY samples were then centrifuged at 15,000 rpm for 10 min at 4°C, and the SP:SAMPLEPREP_SUMMARY supernatant was transferred to 2 mL tubes and dried using a Speed Vac (SPD111V SP:SAMPLEPREP_SUMMARY Savant, Thermo Fisher Scientific). The samples were then reconstituted in SP:SAMPLEPREP_SUMMARY isopropanol:acetonitrile:water (2:1:1 %v/v) and 2.5 μL Splash® Lipidomix® SP:SAMPLEPREP_SUMMARY (Avanti Polar Lipids Inc.) was added to each sample as an internal standard. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The mobile phase involved a gradient comprising varying amounts of solutions A CH:CHROMATOGRAPHY_SUMMARY (10 mM ammonium formate in 60% v/v acetonitrile) and B (10 mM ammonium formate CH:CHROMATOGRAPHY_SUMMARY in 95% isopropanol and 5% acetonitrile). The linear gradient elution was: CH:CHROMATOGRAPHY_SUMMARY 40–43% B (0–2 min), 43–50% B (over 0.1 min), 50–54% B (over 10 min), CH:CHROMATOGRAPHY_SUMMARY 54–70% B (over 0.1 min), 70–99% B (over 5.9 min), 99-40% B (over 0.1 min), CH:CHROMATOGRAPHY_SUMMARY and 40% B (for 2 min), run at 300 μl/min in positive Electrospray Ionization CH:CHROMATOGRAPHY_SUMMARY mode and the column temperature was maintained at 55 °C CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters CH:COLUMN_NAME Waters Acquity BEH C18 (100 x 2.1mm, 1.7um) CH:FLOW_RATE 300 μl/min CH:SOLVENT_A 10 mM ammonium formate in 60% v/v acetonitrile CH:SOLVENT_B 10 mM ammonium formate in 95% isopropanol and 5% acetonitrile CH:INJECTION_TEMPERATURE 4 CH:INTERNAL_STANDARD SPLASH™ Lipidomix® Mass Spec Standard (Avanti Polar Lipids, Inc.) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 XS QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST001093_AN001779_Results.txt UNITS:abundance corresponding to m/z Has m/z:Yes Has RT:Yes RT units:Seconds #END