#METABOLOMICS WORKBENCH bsmith8_20250327_095335 DATATRACK_ID:5796 STUDY_ID:ST003835 ANALYSIS_ID:AN006297 PROJECT_ID:PR002395 VERSION 1 CREATED_ON March 28, 2025, 7:49 am #PROJECT PR:PROJECT_TITLE Metabolomics of wild-type and TauT-/- leukemia cells. PR:PROJECT_SUMMARY We carried out untargeted metabolomic analysis of Lin- leukemia stem cells from PR:PROJECT_SUMMARY mice bearing wild type and TauT-/- leukemias. Briefly, C57BL/6J mice were PR:PROJECT_SUMMARY retro-orbitally transplanted with wild type and TauT-/- leukemia stem cells. The PR:PROJECT_SUMMARY mice were sacrificed on days 11-13. Leukemia cells were isolated and PR:PROJECT_SUMMARY magnetically enriched for lin- leukemia stem cells. Untargeted metabolomic PR:PROJECT_SUMMARY analysis was carried out to identify metabolic changes induced by inhibiting PR:PROJECT_SUMMARY taurine uptake in leukemia cells. PR:INSTITUTE University of Rochester Medical Center PR:LAST_NAME Smith PR:FIRST_NAME Bradley PR:ADDRESS 575 Elmwood Ave, Rochester, NY, 14620 PR:EMAIL bradley_smith@urmc.rochester.edu PR:PHONE 585-275-1445 #STUDY ST:STUDY_TITLE metabolomics of wild-type and TauT-/- leukemia cells ST:STUDY_SUMMARY C57BL/6J mice conditioned with irradiation were transplanted with wild type and ST:STUDY_SUMMARY TauT-/- leukemia stem cells. The mice were sacrificed on days 11-13 ST:STUDY_SUMMARY post-transplant. Spleens from leukemic mice were quickly dissected and ST:STUDY_SUMMARY dissociated in Hanks’ balanced salt solution (Gibco) with 5% fetal bovine ST:STUDY_SUMMARY serum and 2mM EDTA at 4C. The leukemia cells were stained for Lin+ (CD3ε-, ST:STUDY_SUMMARY CD4-, CD8-, Gr1-, CD11b/Mac-1-, TER119-, CD45R/B220- and CD19-) markers and were ST:STUDY_SUMMARY magnetically depleted using LD columns (Milteny Biotec). Lin- leukemia stem cell ST:STUDY_SUMMARY fraction was washed with PBS containing 5mM glucose and centrifuged at 3000g for ST:STUDY_SUMMARY 1 min, snap frozen, and processed for untargeted metabolomics ST:INSTITUTE University of Rochester Medical Center ST:LAST_NAME Smith ST:FIRST_NAME Bradley ST:ADDRESS 575 Elmwood Ave, Rochester, NY, 14620 ST:EMAIL bradley_smith@urmc.rochester.edu ST:PHONE 585-275-1445 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - BcCML_KO_1_17 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_1_240819_17 SUBJECT_SAMPLE_FACTORS - BcCML_KO_2_18 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_2_240819_18 SUBJECT_SAMPLE_FACTORS - BcCML_KO_3_19 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_3_240819_19 SUBJECT_SAMPLE_FACTORS - BcCML_KO_4_20 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_4_240819_20 SUBJECT_SAMPLE_FACTORS - BcCML_KO_5_21 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_5_240819_21 SUBJECT_SAMPLE_FACTORS - BcCML_KO_6_22 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_6_240819_22 SUBJECT_SAMPLE_FACTORS - BcCML_KO_7_23 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_7_240819_23 SUBJECT_SAMPLE_FACTORS - BcCML_KO_8_24 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_8_240819_24 SUBJECT_SAMPLE_FACTORS - BcCML_KO_9_25 Sample source:BcCML cells | Genotype:BcCML_KO RAW_FILE_NAME(Raw_File_Name)=BcCML_KO_9_240819_25 SUBJECT_SAMPLE_FACTORS - BcCML_WT_1_1 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_1_240819_1 SUBJECT_SAMPLE_FACTORS - BcCML_WT_2_2 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_2_240819_2 SUBJECT_SAMPLE_FACTORS - BcCML_WT_3_3 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_3_240819_3 SUBJECT_SAMPLE_FACTORS - BcCML_WT_4_4 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_4_240819_4 SUBJECT_SAMPLE_FACTORS - BcCML_WT_5_5 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_5_240819_5 SUBJECT_SAMPLE_FACTORS - BcCML_WT_6_6 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_6_240819_6 SUBJECT_SAMPLE_FACTORS - BcCML_WT_7_7 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_7_240819_7 SUBJECT_SAMPLE_FACTORS - BcCML_WT_8_8 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_8_240819_8 SUBJECT_SAMPLE_FACTORS - BcCML_WT_9_9 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_9_240819_9 SUBJECT_SAMPLE_FACTORS - BcCML_WT_10_10 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_10_240819_10 SUBJECT_SAMPLE_FACTORS - BcCML_WT_11_11 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_11_240819_11 SUBJECT_SAMPLE_FACTORS - BcCML_WT_12_12 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_12_240819_12 SUBJECT_SAMPLE_FACTORS - BcCML_WT_13_13 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_13_240819_13 SUBJECT_SAMPLE_FACTORS - BcCML_WT_14_14 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_14_240819_14 SUBJECT_SAMPLE_FACTORS - BcCML_WT_15_15 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_15_240819_15 SUBJECT_SAMPLE_FACTORS - BcCML_WT_16_16 Sample source:BcCML cells | Genotype:BcCML_WT RAW_FILE_NAME(Raw_File_Name)=BcCML_WT_16_240819_16 #COLLECTION CO:COLLECTION_SUMMARY : C57BL/6J mice conditioned with irradiation were transplanted with wild type CO:COLLECTION_SUMMARY and TauT-/- leukemia stem cells. The mice were sacrificed on days 11-13 CO:COLLECTION_SUMMARY post-transplant. Spleens from leukemic mice were quickly dissected and CO:COLLECTION_SUMMARY dissociated in Hanks’ balanced salt solution (Gibco) with 5% fetal bovine CO:COLLECTION_SUMMARY serum and 2mM EDTA at 4C. The leukemia cells were stained for Lin+ (CD3ε-, CO:COLLECTION_SUMMARY CD4-, CD8-, Gr1-, CD11b/Mac-1-, TER119-, CD45R/B220- and CD19-) markers and were CO:COLLECTION_SUMMARY magnetically depleted using LD columns (Milteny Biotec). Lin- leukemia stem cell CO:COLLECTION_SUMMARY fraction was washed with PBS containing 5mM glucose and centrifuged at 3000g for CO:COLLECTION_SUMMARY 1 min and snap frozen. CO:SAMPLE_TYPE BcCML cells #TREATMENT TR:TREATMENT_SUMMARY Not applicable #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen cell pellets were resuspended at 2 million cells per 1 ml of 80% MeOH via SP:SAMPLEPREP_SUMMARY vortexing, transferred to -80C for 30min and then regular ice for 30 minutes SP:SAMPLEPREP_SUMMARY with vortexing every 10 minutes. Next, samples were centrifuged at 17,000x g for SP:SAMPLEPREP_SUMMARY 10 minutes and 90% of supernatant was dried down in a vacuum evaporator SP:SAMPLEPREP_SUMMARY (Thermo). Samples were reconstituted in 50% acetonitrile (A955, Fisher SP:SAMPLEPREP_SUMMARY Scientific), at a volume equal to 10% of dried down volume, and transferred to SP:SAMPLEPREP_SUMMARY glass vials for LC/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters XBridge XP BEH Amide (150 mm x 2.1 mm, 2.5 µm) CH:SOLVENT_A 100% water; 10 mM ammonium acetate; 0.1% ammonium hydroxide; 0.1% medronic acid CH:SOLVENT_B 90% acetonitrile/10% water; 10 mM ammonium acetate; 0.1% ammonium hydroxide; CH:SOLVENT_B 0.1% medronic acid CH:FLOW_GRADIENT 0 minutes, 100% B; 2 minutes, 100% B; 3 minutes, 90% B; 5 minutes, 90% CH:FLOW_GRADIENT B; 6 minutes, 85% B; 7 minutes, 85% B; 8 minutes, 75% B; 9 minutes, 75% CH:FLOW_GRADIENT B; 10 minutes, 55% B; 12 minutes, 55% B; 13 minutes, 35%, 20 minutes, CH:FLOW_GRADIENT 35% B; 20.1 minutes, 35% B; 20.6 minutes, 100% B; 22.2 minutes, 100% B CH:FLOW_RATE 150 μl min−1 for first 22.7 minutes, then 150 μl min−1 for 22.7 to 28 CH:FLOW_RATE minutes CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Data acquisition software: XCalibur Data processing and feature assignment MS:MS_COMMENTS software: Compound Discoverer MS:MS_RESULTS_FILE ST003835_AN006297_Results.txt UNITS:area Has m/z:Yes Has RT:Yes RT units:Minutes #END