#METABOLOMICS WORKBENCH jli_20210217_034113 DATATRACK_ID:2491 STUDY_ID:ST001707 ANALYSIS_ID:AN002781 PROJECT_ID:PR001092 VERSION 1 CREATED_ON February 22, 2021, 2:18 pm #PROJECT PR:PROJECT_TITLE Lipid Profiling of Mouse Intestinal Organoids for studying APC Mutations PR:PROJECT_SUMMARY Inactivating mutations including both germline and somatic mutations in the PR:PROJECT_SUMMARY adenomatous polyposis coli (APC) gene drives most familial and sporadic PR:PROJECT_SUMMARY colorectal cancers. Understanding the metabolic implications of this mutation PR:PROJECT_SUMMARY will aid to establish its wider impact on cellular behaviour and potentially PR:PROJECT_SUMMARY inform clinical decisions. However, to date, alterations in lipid metabolism PR:PROJECT_SUMMARY induced by APC mutations remain unclear. Intestinal organoids have gained PR:PROJECT_SUMMARY widespread popularity in studying colorectal cancer and chemotherapies, because PR:PROJECT_SUMMARY their three-dimensional structure more accurately mimics an in vivo environment. PR:PROJECT_SUMMARY Here, we aimed to investigate intra-cellular lipid disturbances induced by APC PR:PROJECT_SUMMARY gene mutations in intestinal organoids using a reversed-phase PR:PROJECT_SUMMARY ultra-high-performance liquid chromatography mass spectrometry PR:PROJECT_SUMMARY (RP-UHPLC-MS)-based lipid profiling method. Lipids of the organoids grown from PR:PROJECT_SUMMARY either wildtype (WT) or mice with Apc mutations (Lgr5–EGFP-IRES-CreERT2 PR:PROJECT_SUMMARY Apcfl/fl) were extracted and analysed using RP-UHPLC-MS. Concentrations of PR:PROJECT_SUMMARY phospholipids (e.g. PC(16:0/16:0), PC(18:1/20:0), PC(38:0), PC(18:1/22:1)), PR:PROJECT_SUMMARY ceramides (e.g. Cer(d18:0/22:0), Cer(d42:0), Cer(d18:1/24:1)) and PR:PROJECT_SUMMARY hexosylceramide (e.g. HexCer(d18:1/16:0), HexCer(d18:1/22:0)) were higher in PR:PROJECT_SUMMARY Apcfl/fl organoids, whereas levels of sphingomyelins (e.g. SM(d18:1/14:0), PR:PROJECT_SUMMARY SM(d18:1/16:0) ) were lower compared to WT. These observations indicate that PR:PROJECT_SUMMARY cellular metabolism of sphingomyelin was upregulated, resulting in the cellular PR:PROJECT_SUMMARY accumulation of ceramides and production of HexCer due to the absence of PR:PROJECT_SUMMARY Apcfl/fl in the organoids. Our observations demonstrated lipid profiling of PR:PROJECT_SUMMARY organoids and provided an enhanced insight into the effects of the APC mutations PR:PROJECT_SUMMARY on lipid metabolism, making for a valuable addition to screening options of the PR:PROJECT_SUMMARY organoid lipidome. PR:INSTITUTE Imperial College London PR:LAST_NAME Li PR:FIRST_NAME Jia PR:ADDRESS Imperial College London Road, South Kensington, London, London, SW7 2AZ, United PR:ADDRESS Kingdom PR:EMAIL jia.li@imperial.ac.uk PR:PHONE 02075943230 #STUDY ST:STUDY_TITLE Lipid Profiling of Mouse Intestinal Organoids for studying APC Mutations ST:STUDY_SUMMARY Inactivating mutations including both germline and somatic mutations in the ST:STUDY_SUMMARY adenomatous polyposis coli (APC) gene drives most familial and sporadic ST:STUDY_SUMMARY colorectal cancers. Understanding the metabolic implications of this mutation ST:STUDY_SUMMARY will aid to establish its wider impact on cellular behaviour and potentially ST:STUDY_SUMMARY inform clinical decisions. However, to date, alterations in lipid metabolism ST:STUDY_SUMMARY induced by APC mutations remain unclear. Intestinal organoids have gained ST:STUDY_SUMMARY widespread popularity in studying colorectal cancer and chemotherapies, because ST:STUDY_SUMMARY their three-dimensional structure more accurately mimics an in vivo environment. ST:STUDY_SUMMARY Here, we aimed to investigate intra-cellular lipid disturbances induced by APC ST:STUDY_SUMMARY gene mutations in intestinal organoids using a reversed-phase ST:STUDY_SUMMARY ultra-high-performance liquid chromatography mass spectrometry ST:STUDY_SUMMARY (RP-UHPLC-MS)-based lipid profiling method. Lipids of the organoids grown from ST:STUDY_SUMMARY either wildtype (WT) or mice with Apc mutations (Lgr5–EGFP-IRES-CreERT2 ST:STUDY_SUMMARY Apcfl/fl) were extracted and analysed using RP-UHPLC-MS. Concentrations of ST:STUDY_SUMMARY phospholipids (e.g. PC(16:0/16:0), PC(18:1/20:0), PC(38:0), PC(18:1/22:1)), ST:STUDY_SUMMARY ceramides (e.g. Cer(d18:0/22:0), Cer(d42:0), Cer(d18:1/24:1)) and ST:STUDY_SUMMARY hexosylceramide (e.g. HexCer(d18:1/16:0), HexCer(d18:1/22:0)) were higher in ST:STUDY_SUMMARY Apcfl/fl organoids, whereas levels of sphingomyelins (e.g. SM(d18:1/14:0), ST:STUDY_SUMMARY SM(d18:1/16:0) ) were lower compared to WT. These observations indicate that ST:STUDY_SUMMARY cellular metabolism of sphingomyelin was upregulated, resulting in the cellular ST:STUDY_SUMMARY accumulation of ceramides and production of HexCer due to the absence of ST:STUDY_SUMMARY Apcfl/fl in the organoids. Our observations demonstrated lipid profiling of ST:STUDY_SUMMARY organoids and provided an enhanced insight into the effects of the APC mutations ST:STUDY_SUMMARY on lipid metabolism, making for a valuable addition to screening options of the ST:STUDY_SUMMARY organoid lipidome. ST:INSTITUTE Imperial College London ST:LAST_NAME Li ST:FIRST_NAME Jia ST:ADDRESS Imperial College London, UK ST:EMAIL jia.li@imperial.ac.uk ST:PHONE 00442075943230 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #FACTORS #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 m1641 NEG_WT_m1641_1 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1641_1; sample replicate IDs=1; MS mode=negative SUBJECT_SAMPLE_FACTORS m1641 NEG_WT_m1641_2 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1641_2; sample replicate IDs=2; MS mode=negative SUBJECT_SAMPLE_FACTORS m1641 NEG_WT_m1641_3 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1641_3; sample replicate IDs=3; MS mode=negative SUBJECT_SAMPLE_FACTORS m1641 NEG_WT_m1641_4 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1641_4; sample replicate IDs=4; MS mode=negative SUBJECT_SAMPLE_FACTORS m1641 NEG_WT_m1641_5 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1641_5; sample replicate IDs=5; MS mode=negative SUBJECT_SAMPLE_FACTORS m1641 NEG_WT_m1641_6 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1641_6; sample replicate IDs=6; MS mode=negative SUBJECT_SAMPLE_FACTORS m1641 NEG_WT_m1641_7 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1641_7; sample replicate IDs=7; MS mode=negative SUBJECT_SAMPLE_FACTORS m1641 NEG_WT_m1641_8 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1641_8; sample replicate IDs=8; MS mode=negative SUBJECT_SAMPLE_FACTORS m1663 NEG_WT_m1663_1 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1663_1; sample replicate IDs=1; MS mode=negative SUBJECT_SAMPLE_FACTORS m1663 NEG_WT_m1663_2 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1663_2; sample replicate IDs=2; MS mode=negative SUBJECT_SAMPLE_FACTORS m1663 NEG_WT_m1663_3 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1663_3; sample replicate IDs=3; MS mode=negative SUBJECT_SAMPLE_FACTORS m1663 NEG_WT_m1663_4 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1663_4; sample replicate IDs=4; MS mode=negative SUBJECT_SAMPLE_FACTORS m1663 NEG_WT_m1663_5 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1663_5; sample replicate IDs=5; MS mode=negative SUBJECT_SAMPLE_FACTORS m1663 NEG_WT_m1663_6 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1663_6; sample replicate IDs=6; MS mode=negative SUBJECT_SAMPLE_FACTORS m1663 NEG_WT_m1663_7 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1663_7; sample replicate IDs=7; MS mode=negative SUBJECT_SAMPLE_FACTORS m1663 NEG_WT_m1663_8 Genotype:wildtype RAW_FILE_NAME=NEG_WT_m1663_8; sample replicate IDs=8; MS mode=negative SUBJECT_SAMPLE_FACTORS m830 NEG_APC_m830_1 Genotype:APC RAW_FILE_NAME=NEG_APC_m830_1; sample replicate IDs=1; MS mode=negative SUBJECT_SAMPLE_FACTORS m830 NEG_APC_m830_2 Genotype:APC RAW_FILE_NAME=NEG_APC_m830_2; sample replicate IDs=2; MS mode=negative SUBJECT_SAMPLE_FACTORS m830 NEG_APC_m830_3 Genotype:APC RAW_FILE_NAME=NEG_APC_m830_3; sample replicate IDs=3; MS mode=negative SUBJECT_SAMPLE_FACTORS m830 NEG_APC_m830_4 Genotype:APC RAW_FILE_NAME=NEG_APC_m830_4; sample replicate IDs=4; MS mode=negative SUBJECT_SAMPLE_FACTORS m830 NEG_APC_m830_5 Genotype:APC RAW_FILE_NAME=NEG_APC_m830_5; sample replicate IDs=5; MS mode=negative SUBJECT_SAMPLE_FACTORS m830 NEG_APC_m830_6 Genotype:APC RAW_FILE_NAME=NEG_APC_m830_6; sample replicate IDs=6; MS mode=negative SUBJECT_SAMPLE_FACTORS m830 NEG_APC_m830_7 Genotype:APC RAW_FILE_NAME=NEG_APC_m830_7; sample replicate IDs=7; MS mode=negative SUBJECT_SAMPLE_FACTORS m830 NEG_APC_m830_8 Genotype:APC RAW_FILE_NAME=NEG_APC_m830_8; sample replicate IDs=8; MS mode=negative SUBJECT_SAMPLE_FACTORS m862 NEG_APC_m862_1 Genotype:APC RAW_FILE_NAME=NEG_APC_m862_1; sample replicate IDs=1; MS mode=negative SUBJECT_SAMPLE_FACTORS m862 NEG_APC_m862_2 Genotype:APC RAW_FILE_NAME=NEG_APC_m862_2; sample replicate IDs=2; MS mode=negative SUBJECT_SAMPLE_FACTORS m862 NEG_APC_m862_3 Genotype:APC RAW_FILE_NAME=NEG_APC_m862_3; sample replicate IDs=3; MS mode=negative SUBJECT_SAMPLE_FACTORS m862 NEG_APC_m862_4 Genotype:APC RAW_FILE_NAME=NEG_APC_m862_4; sample replicate IDs=4; MS mode=negative SUBJECT_SAMPLE_FACTORS m862 NEG_APC_m862_5 Genotype:APC RAW_FILE_NAME=NEG_APC_m862_5; sample replicate IDs=5; MS mode=negative SUBJECT_SAMPLE_FACTORS m862 NEG_APC_m862_6 Genotype:APC RAW_FILE_NAME=NEG_APC_m862_6; sample replicate IDs=6; MS mode=negative SUBJECT_SAMPLE_FACTORS m862 NEG_APC_m862_7 Genotype:APC RAW_FILE_NAME=NEG_APC_m862_7; sample replicate IDs=7; MS mode=negative SUBJECT_SAMPLE_FACTORS m862 NEG_APC_m862_8 Genotype:APC RAW_FILE_NAME=NEG_APC_m862_8; sample replicate IDs=8; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control NEG_QC1 Genotype:quality control RAW_FILE_NAME=NEG_QC1; sample replicate IDs=quality control; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control NEG_QC2 Genotype:quality control RAW_FILE_NAME=NEG_QC2; sample replicate IDs=quality control; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control NEG_QC3 Genotype:quality control RAW_FILE_NAME=NEG_QC3; sample replicate IDs=quality control; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control NEG_QC4 Genotype:quality control RAW_FILE_NAME=NEG_QC4; sample replicate IDs=quality control; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control NEG_QC5 Genotype:quality control RAW_FILE_NAME=NEG_QC5; sample replicate IDs=quality control; MS mode=negative SUBJECT_SAMPLE_FACTORS blank NEG_Blank Genotype:blank RAW_FILE_NAME=NEG_Blank; sample replicate IDs=blank; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_2_1 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_2_1; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_2_2 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_2_2; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_2_3 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_2_3; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_4_1 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_4_1; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_4_2 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_4_2; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_4_3 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_4_3; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_8_1 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_8_1; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_8_2 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_8_2; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS quality control dilutions NEG_Dilution1_8_3 Genotype:quality control dilutions RAW_FILE_NAME=NEG_Dilution1_8_3; sample replicate IDs=quality control dilutions; MS mode=negative SUBJECT_SAMPLE_FACTORS m1641 POS_WT_m1641_1 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1641_1; sample replicate IDs=1; MS mode=positive SUBJECT_SAMPLE_FACTORS m1641 POS_WT_m1641_2 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1641_2; sample replicate IDs=2; MS mode=positive SUBJECT_SAMPLE_FACTORS m1641 POS_WT_m1641_3 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1641_3; sample replicate IDs=3; MS mode=positive SUBJECT_SAMPLE_FACTORS m1641 POS_WT_m1641_4 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1641_4; sample replicate IDs=4; MS mode=positive SUBJECT_SAMPLE_FACTORS m1641 POS_WT_m1641_5 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1641_5; sample replicate IDs=5; MS mode=positive SUBJECT_SAMPLE_FACTORS m1641 POS_WT_m1641_6 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1641_6; sample replicate IDs=6; MS mode=positive SUBJECT_SAMPLE_FACTORS m1641 POS_WT_m1641_7 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1641_7; sample replicate IDs=7; MS mode=positive SUBJECT_SAMPLE_FACTORS m1641 POS_WT_m1641_8 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1641_8; sample replicate IDs=8; MS mode=positive SUBJECT_SAMPLE_FACTORS m1663 POS_WT_m1663_1 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1663_1; sample replicate IDs=1; MS mode=positive SUBJECT_SAMPLE_FACTORS m1663 POS_WT_m1663_2 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1663_2; sample replicate IDs=2; MS mode=positive SUBJECT_SAMPLE_FACTORS m1663 POS_WT_m1663_3 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1663_3; sample replicate IDs=3; MS mode=positive SUBJECT_SAMPLE_FACTORS m1663 POS_WT_m1663_4 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1663_4; sample replicate IDs=4; MS mode=positive SUBJECT_SAMPLE_FACTORS m1663 POS_WT_m1663_5 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1663_5; sample replicate IDs=5; MS mode=positive SUBJECT_SAMPLE_FACTORS m1663 POS_WT_m1663_6 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1663_6; sample replicate IDs=6; MS mode=positive SUBJECT_SAMPLE_FACTORS m1663 POS_WT_m1663_7 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1663_7; sample replicate IDs=7; MS mode=positive SUBJECT_SAMPLE_FACTORS m1663 POS_WT_m1663_8 Genotype:wildtype RAW_FILE_NAME=POS_WT_m1663_8; sample replicate IDs=8; MS mode=positive SUBJECT_SAMPLE_FACTORS m830 POS_APC_m830_1 Genotype:APC RAW_FILE_NAME=POS_APC_m830_1; sample replicate IDs=1; MS mode=positive SUBJECT_SAMPLE_FACTORS m830 POS_APC_m830_2 Genotype:APC RAW_FILE_NAME=POS_APC_m830_2; sample replicate IDs=2; MS mode=positive SUBJECT_SAMPLE_FACTORS m830 POS_APC_m830_3 Genotype:APC RAW_FILE_NAME=POS_APC_m830_3; sample replicate IDs=3; MS mode=positive SUBJECT_SAMPLE_FACTORS m830 POS_APC_m830_4 Genotype:APC RAW_FILE_NAME=POS_APC_m830_4; sample replicate IDs=4; MS mode=positive SUBJECT_SAMPLE_FACTORS m830 POS_APC_m830_5 Genotype:APC RAW_FILE_NAME=POS_APC_m830_5; sample replicate IDs=5; MS mode=positive SUBJECT_SAMPLE_FACTORS m830 POS_APC_m830_6 Genotype:APC RAW_FILE_NAME=POS_APC_m830_6; sample replicate IDs=6; MS mode=positive SUBJECT_SAMPLE_FACTORS m830 POS_APC_m830_7 Genotype:APC RAW_FILE_NAME=POS_APC_m830_7; sample replicate IDs=7; MS mode=positive SUBJECT_SAMPLE_FACTORS m830 POS_APC_m830_8 Genotype:APC RAW_FILE_NAME=POS_APC_m830_8; sample replicate IDs=8; MS mode=positive SUBJECT_SAMPLE_FACTORS m862 POS_APC_m862_1 Genotype:APC RAW_FILE_NAME=POS_APC_m862_1; sample replicate IDs=1; MS mode=positive SUBJECT_SAMPLE_FACTORS m862 POS_APC_m862_2 Genotype:APC RAW_FILE_NAME=POS_APC_m862_2; sample replicate IDs=2; MS mode=positive SUBJECT_SAMPLE_FACTORS m862 POS_APC_m862_3 Genotype:APC RAW_FILE_NAME=POS_APC_m862_3; sample replicate IDs=3; MS mode=positive SUBJECT_SAMPLE_FACTORS m862 POS_APC_m862_4 Genotype:APC RAW_FILE_NAME=POS_APC_m862_4; sample replicate IDs=4; MS mode=positive SUBJECT_SAMPLE_FACTORS m862 POS_APC_m862_5 Genotype:APC RAW_FILE_NAME=POS_APC_m862_5; sample replicate IDs=5; MS mode=positive SUBJECT_SAMPLE_FACTORS m862 POS_APC_m862_6 Genotype:APC RAW_FILE_NAME=POS_APC_m862_6; sample replicate IDs=6; MS mode=positive SUBJECT_SAMPLE_FACTORS m862 POS_APC_m862_7 Genotype:APC RAW_FILE_NAME=POS_APC_m862_7; sample replicate IDs=7; MS mode=positive SUBJECT_SAMPLE_FACTORS m862 POS_APC_m862_8 Genotype:APC RAW_FILE_NAME=POS_APC_m862_8; sample replicate IDs=8; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control POS_QC1 Genotype:quality control RAW_FILE_NAME=POS_QC1; sample replicate IDs=quality control; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control POS_QC2 Genotype:quality control RAW_FILE_NAME=POS_QC2; sample replicate IDs=quality control; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control POS_QC3 Genotype:quality control RAW_FILE_NAME=POS_QC3; sample replicate IDs=quality control; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control POS_QC4 Genotype:quality control RAW_FILE_NAME=POS_QC4; sample replicate IDs=quality control; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control POS_QC5 Genotype:quality control RAW_FILE_NAME=POS_QC5; sample replicate IDs=quality control; MS mode=positive SUBJECT_SAMPLE_FACTORS blank POS_Blank Genotype:blank RAW_FILE_NAME=POS_Blank; sample replicate IDs=blank; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_2_1 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_2_1; sample replicate IDs=quality control dilutions; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_2_2 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_2_2; sample replicate IDs=quality control dilutions; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_2_3 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_2_3; sample replicate IDs=quality control dilutions; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_4_1 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_4_1; sample replicate IDs=quality control dilutions; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_4_2 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_4_2; sample replicate IDs=quality control dilutions; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_4_3 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_4_3; sample replicate IDs=quality control dilutions; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_8_1 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_8_1; sample replicate IDs=quality control dilutions; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_8_2 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_8_2; sample replicate IDs=quality control dilutions; MS mode=positive SUBJECT_SAMPLE_FACTORS quality control dilutions POS_Dilution1_8_3 Genotype:quality control dilutions RAW_FILE_NAME=POS_Dilution1_8_3; sample replicate IDs=quality control dilutions; MS mode=positive #COLLECTION CO:COLLECTION_SUMMARY Control (Lgr5–EGFP-IRES-CreERT2 Apc+/+) or experimental CO:COLLECTION_SUMMARY (Lgr5–EGFP-IRES-CreERT2 Apcfl/fl) adult mice were administered tamoxifen (80 CO:COLLECTION_SUMMARY mg/kg) daily via intraperitoneal injection for 4 consecutive days to induce Cre CO:COLLECTION_SUMMARY expression. Fourteen days following induction mice were sacrificed (cervical CO:COLLECTION_SUMMARY dislocation) and their intestinal cells were harvested for organoid culture of CO:COLLECTION_SUMMARY WT or Apc deficient intestinal stem cells. CO:SAMPLE_TYPE Intestine #TREATMENT TR:TREATMENT_SUMMARY no treatment was applied #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Following aqueous extraction using cold methanol and water (v:v, 1:1), 1 ml of SP:SAMPLEPREP_SUMMARY pre-chilled dichloromethane (DCM)/methanol (v:v, 3:1) was added to the organoid SP:SAMPLEPREP_SUMMARY samples. Samples were bead-beaten for 40 seconds followed by five minutes of SP:SAMPLEPREP_SUMMARY chilling on dry ice. This procedure was repeated three times before being SP:SAMPLEPREP_SUMMARY centrifuged for 10 mins at 21,000 rcf at 4ºC. A total of 600 μl of supernatant SP:SAMPLEPREP_SUMMARY from each sample was transferred to a glass vial. Another 200 μl of supernatant SP:SAMPLEPREP_SUMMARY from each sample was pooled into a 15-ml Falcon tube to form a quantity control SP:SAMPLEPREP_SUMMARY (QC) sample and split into several aliquots of 600 μl each. An extraction blank SP:SAMPLEPREP_SUMMARY sample was included to control for any potential contaminant introduced SP:SAMPLEPREP_SUMMARY throughout the extraction process. Samples were dried by evaporation over night SP:SAMPLEPREP_SUMMARY at room temperature and stored at -40˚C until further analysis. The dried SP:SAMPLEPREP_SUMMARY extracts were reconstituted in 100 μl of water/acetonitrile (ACN)/isopropanol SP:SAMPLEPREP_SUMMARY (IPA), (v:v:v, 1:1:3,). The lipids were dissolved by vigorous vortexing for five SP:SAMPLEPREP_SUMMARY minutes, followed by five minutes of sonication. This step was repeated three SP:SAMPLEPREP_SUMMARY times to allow the dry extracts to thoroughly dissolve in the solvent. Samples SP:SAMPLEPREP_SUMMARY were subsequently centrifuged at 21,000 rcf for 10 minutes at 4ºC and SP:SAMPLEPREP_SUMMARY transferred to 150-μl glass inserts placed in glass vials (Waters). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Acquity UPLC CH:COLUMN_NAME Waters Acquity C18 CSH (2.1 x 10 mm, 1.7 μm) #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE jli_20210217_034113_PR_MS_Methodology.docx #MS MS:INSTRUMENT_NAME Waters Synapt G2 S QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS refer to Methodology.docx MS:MS_RESULTS_FILE ST001707_AN002781_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END