#METABOLOMICS WORKBENCH vanessa_mancini_20200829_105246 DATATRACK_ID:2154 STUDY_ID:ST001494 ANALYSIS_ID:AN002477 PROJECT_ID:PR001012 VERSION 1 CREATED_ON September 29, 2020, 2:07 pm #PROJECT PR:PROJECT_TITLE Embryo device MS study PR:PROJECT_SUMMARY Metabolomics study of murine embryos cultured in an innovative microfluidic PR:PROJECT_SUMMARY device to assess release of plastic-related compounds and embryo metabolic PR:PROJECT_SUMMARY activity. PR:INSTITUTE University of Leeds PR:DEPARTMENT Electronic and Electrical Engineering PR:LABORATORY Bioelectronics Lab PR:LAST_NAME Mancini PR:FIRST_NAME Vanessa PR:ADDRESS Woodhouse Lane, Leeds, West Yorkshire, LS62HN, United Kingdom PR:EMAIL elvm@leeds.ac.uk PR:PHONE +447599197366 #STUDY ST:STUDY_TITLE Metabolomics of murine embryos cultured in a microfluidic device and comparison ST:STUDY_TITLE with traditional microdrops culture ST:STUDY_SUMMARY Global untargeted metabolomics study to analyse culture media extracted from an ST:STUDY_SUMMARY innovative microfluidic device or traditional microdrops in presence or absence ST:STUDY_SUMMARY of murine embryos to investigate PDMS-release of biomolecules and embryo ST:STUDY_SUMMARY metabolic activity. ST:INSTITUTE University of Leeds ST:LAST_NAME Mancini ST:FIRST_NAME Vanessa ST:ADDRESS Woodhouse Lane, Leeds, LS29JT ST:EMAIL elvm@leeds.ac.uk ST:PHONE +447599197366 #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 - SC_20190605_RPLCp_FMS_Embryo_S1b Device:Control_device Group name=Day 0 KSOM SUBJECT_SAMPLE_FACTORS - SC_20190605_RPLCp_FMS_Embryo_S2b Device:Control_device Group name=Day 0 KSOM SUBJECT_SAMPLE_FACTORS - SC_20190605_RPLCp_FMS_Embryo_S3b Device:Control_device Group name=Day 0 KSOM SUBJECT_SAMPLE_FACTORS - SC_20190605_RPLCp_FMS_Embryo_S7 Device:No embryos_device Group name=Day 5 KSOM_device SUBJECT_SAMPLE_FACTORS - SC_20190605_RPLCp_FMS_Embryo_S8 Device:No embryos_device Group name=Day 5 KSOM_device SUBJECT_SAMPLE_FACTORS - SC_20190605_RPLCp_FMS_Embryo_S9 Device:No embryos_device Group name=Day 5 KSOM_device SUBJECT_SAMPLE_FACTORS - SC_20190605_RPLCp_FMS_Embryo_S13 Device:Embryo culture media_device Group name=Day 5 KSOM_device_embryos SUBJECT_SAMPLE_FACTORS - SC_20190605_RPLCp_FMS_Embryo_S14 Device:Embryo culture media_device Group name=Day 5 KSOM_device_embryos SUBJECT_SAMPLE_FACTORS - SC_20190605_RPLCp_FMS_Embryo_S15 Device:Embryo culture media_device Group name=Day 5 KSOM_device_embryos SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S01_T2 Device:Control_drops Group name=Day 0 KSOM SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S02_T1 Device:Control_drops Group name=Day 0 KSOM SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S03_T1 Device:Control_drops Group name=Day 0 KSOM SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S07_T1 Device:No embryos_drops Group name=Day 4 KSOM_drop SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S08_T1 Device:No embryos_drops Group name=Day 4 KSOM_drop SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S09_T2 Device:No embryos_drops Group name=Day 4 KSOM_drop SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S13_T1 Device:Embryo culture media_drops Group name=Day 4 KSOM_drop_embryos SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S14_T2 Device:Embryo culture media_drops Group name=Day 4 KSOM_drop_embryos SUBJECT_SAMPLE_FACTORS - SC_20191202_FMS_Embryo_S15_T1 Device:Embryo culture media_drops Group name=Day 4 KSOM_drop_embryos #COLLECTION CO:COLLECTION_SUMMARY Samples were collected from microdrops and microfluidic devices when embryos CO:COLLECTION_SUMMARY developed to fully expanded blastocyst stage, to allow stage-matched comparison CO:COLLECTION_SUMMARY of embryo metabolite production/consumption. CO:SAMPLE_TYPE Blastocysts #TREATMENT TR:TREATMENT_SUMMARY Control KSOM: fresh medium at day 0 not incubated in devices or microdrops. Day TR:TREATMENT_SUMMARY 4 KSOM_drop: medium incubated in microdrops for 4 days without embryos Day 5 TR:TREATMENT_SUMMARY KSOM_device: medium incubated in devices for 5 days without embryos Day 4 TR:TREATMENT_SUMMARY KSOM_drop_embryos: medium incubated in microdrops for 4 days with embryos Day 5 TR:TREATMENT_SUMMARY KSOM_device_embryos: medium incubated in devices for 5 days with embryos #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Culture media samples (100 µl) were thawed on ice and prepared using previously SP:SAMPLEPREP_SUMMARY described methods. Briefly, 300 µL of dry ice cooled methanol was added to SP:SAMPLEPREP_SUMMARY individual culture medium samples and incubated overnight at -80°C; individual SP:SAMPLEPREP_SUMMARY samples were spun down to remove proteins and subsequent supernatant used for SP:SAMPLEPREP_SUMMARY analyses. Samples were separated and analysed using reverse-phase liquid SP:SAMPLEPREP_SUMMARY chromatography connected to a a Thermo Scientific Q Exactive HF (LC-Hybrid SP:SAMPLEPREP_SUMMARY Quadrupole-Orbitrap MS/MS) instrument using positive ion mode MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Hypersil Gold (1.9 µm, 2.1mm x 100 mm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Full MS analyses were acquired over a mass range of m/z 70-1050 under an ESI MS:MS_COMMENTS positive profile mode. Full mass scan was used at a resolution of 120,000 with a MS:MS_COMMENTS scan rate at ∼3.5 Hz Raw data were imported, processed, normalized, and MS:MS_COMMENTS reviewed using Progenesis QI v.2.1 (Non-linear Dynamics, Newcastle, UK). All FMS MS:MS_COMMENTS sample runs were aligned against a FMS QC pool reference, with alignment to the MS:MS_COMMENTS reference being ≥ 96%, demonstrating the reproducibility of the RPLC column MS:MS_COMMENTS separation method. Peak picking, with a minimum threshold of 100,000 ion MS:MS_COMMENTS intensity, was performed for individual aligned runs based on an aggregate run MS:MS_COMMENTS (representative of all ion peaks detected in all samples). MS:MS_RESULTS_FILE ST001494_AN002477_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END