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MB Sample ID: SA191225

Local Sample ID:	RS-01683157
Subject ID:	SU002112
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002112
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
RS-01683157	SA191225	FL023456	Pre-intervention	Treatment

Collection:

Collection ID:	CO002105
Collection Summary:	Samples were taken before and after 6 weeks of flaxseed dietary intervention. Serum and fecal metabolomics were performed to characterize the metabolomic profiles of serum and stool metabolites pre- and post-intervention.
Sample Type:	Feces

Treatment:

Treatment ID:	TR002124
Treatment Summary:	Samples were taken before and after 6 weeks of flaxseed dietary intervention. Serum and fecal metabolomics were performed to characterize the metabolomic profiles of serum and stool metabolites pre- and post-intervention.

Sample Preparation:

Sampleprep ID:	SP002118
Sampleprep Summary:	Different procedures have to be used for different sample matrices; this SOP focuses mainly on bile acids in blood plasma but includes other current protocols 5.1. Isolation of an enriched bile acid fraction from blood plasma or serum • Prepare a plate map showing location of samples in wells. Include wells with method blanks and controls on each plate. • Per well add 50µL plasma sample (excluding Method Blanks, IS Check and Utak Controls) -Method Blanks: Contains Anti-Oxidant Solution, CUDA/PHAU in MeOH:ACN Final Vol 250uL -IS Check: Contains Anti-Oxidant Solution, SSTD, CUDA/PHAU in MeOH:ACN Final Vol 250uL -Utak Controls: Conatins 50uL Utak Serum, Anti-Oxidant Sol, SSTD, CUDA/PAHU in MeOH:ACN Final Vol 250uL ● To each well, add 25 µL Anti-Oxidant Solution ● To each IS Check, Utak Control and Customer Sample, add 25 µL SSTD (Final Volume 100nM) ● To each well, add 25 µL CUDA/PHAU (Final Volume 100nM) ● Add 50:50 MeOH:ACN to each well for a final volume of 250uL.Cap or seal plate with Silicon or Foil Mat and vortex for 1 minute at speed 7. ● Centrifuge plate for 5 minutes so protein precipitates into firm pellet at the bottom of each well ● Pipette roughly 200uL of supernatant into 96 Well PVDF Filter Plate with additional 96 Well Nunc Sample plate ● Centrifuge PVDF Filter plate containing samples into collection plate ● Cover plate with Silicon Nunc Plug mat if plate is being stored at -20C, Cover with Pre-Slit Mat for LC-MS Analysis 5.2. Isolation of an enriched bile acid fraction from tissue, e.g. liver or spinal cord ● Weigh tissue sample into eppendorf tube (amount depending on bile acid content) ● Add 10uL Anti-Oxidant Solution (0.2 mg/ml solution BHT/EDTA in 1:1 methanol/water) use repeater pipetter or syringe repeater assembly. ● Add 10uL SSTD 1000nM Solution, use repeater pipetter or syringe repeater assembly ● Add 500uL Cold MeOH and 2 steel balls (3 mm diameter) and homogenize with GenoGrinder in two 30 sec intervals at 1500 rpm until a fine suspension is achieved. ● Centrifuge at 2C for 3 minutes spinning at 15,000rcf. Transfer methanol supernatant to 1.5 mL eppendorf vial containing 10uL 20% glycerol solution ● Add 500uL Cold MeOH and repeat homogenization with GenoGrinder in two 30 sec intervals at 1500 rpm until a fine suspension is achieved. Centrifuge again and add to the same 1.5mL tube ● Evaporated to dryness, or if a large number of samples has to be processed to a 96-well plate ● Re-suspend pellet in 100uL CUDA/PHAU 100nM MeOH:ACN ● Shake reconstitution for 1 minute at speed of 7 and let stand on wet ice for 15 minutes protected from light ● Shake reconstitution again and filter using PVDF Filter plate or Durapore Spin Filters. ● Transfer filtrate to glass insert in amber vial and cap or 96 well plate with pre-slit silicon mat ● Store at -20°C until LCMS analysis 5.3. Isolation of an enriched bile acid fraction from fecal water ● Label eppendorf tubes; add additional vials for blanks and controls ● Add 10uL Anti-Oxidant Solution using repeater pipetter or syringe repeater assembly ● Add 10uL SSTD using repeater pipetter or syringe repeater assembly ● Thaw out samples of fecal water, vortex briefly to homogenize, keep on ice ● Pipette 10uL of fecal water sample into labeled vials. ● Add 800uL of cold extraction solvent, acetonitrile containing 5% of ammonia ● Vortex samples, then shake for 6 minutes at 4 degree C ● Centrifuge for 3 minutes ● Transfer supernatant to fresh 1.5 mL eppendorf vials and evaporated to dryness, or for a large number of samples, to a 96-well plate and discard centrifugation residues ● Add 100µL CUDA/PHAU IS 100nM to dried sample, cap and vortex 10 sec to dissolve residues ● Set rack of samples on wet ice for 15min ● Filter using PVDF Plate or individual Spin Filters ● Transfer filtrates to glass insert in amber vial and cap or to 96 Well Plate ● Store at -20°C until LCMS analysis

Sampleprep ID:

SP002118

Sampleprep Summary:

Different procedures have to be used for different sample matrices; this SOP focuses mainly on bile acids in blood plasma but includes other current protocols 5.1. Isolation of an enriched bile acid fraction from blood plasma or serum • Prepare a plate map showing location of samples in wells. Include wells with method blanks and controls on each plate. • Per well add 50µL plasma sample (excluding Method Blanks, IS Check and Utak Controls) -Method Blanks: Contains Anti-Oxidant Solution, CUDA/PHAU in MeOH:ACN Final Vol 250uL -IS Check: Contains Anti-Oxidant Solution, SSTD, CUDA/PHAU in MeOH:ACN Final Vol 250uL -Utak Controls: Conatins 50uL Utak Serum, Anti-Oxidant Sol, SSTD, CUDA/PAHU in MeOH:ACN Final Vol 250uL ● To each well, add 25 µL Anti-Oxidant Solution ● To each IS Check, Utak Control and Customer Sample, add 25 µL SSTD (Final Volume 100nM) ● To each well, add 25 µL CUDA/PHAU (Final Volume 100nM) ● Add 50:50 MeOH:ACN to each well for a final volume of 250uL.Cap or seal plate with Silicon or Foil Mat and vortex for 1 minute at speed 7. ● Centrifuge plate for 5 minutes so protein precipitates into firm pellet at the bottom of each well ● Pipette roughly 200uL of supernatant into 96 Well PVDF Filter Plate with additional 96 Well Nunc Sample plate ● Centrifuge PVDF Filter plate containing samples into collection plate ● Cover plate with Silicon Nunc Plug mat if plate is being stored at -20C, Cover with Pre-Slit Mat for LC-MS Analysis 5.2. Isolation of an enriched bile acid fraction from tissue, e.g. liver or spinal cord ● Weigh tissue sample into eppendorf tube (amount depending on bile acid content) ● Add 10uL Anti-Oxidant Solution (0.2 mg/ml solution BHT/EDTA in 1:1 methanol/water) use repeater pipetter or syringe repeater assembly. ● Add 10uL SSTD 1000nM Solution, use repeater pipetter or syringe repeater assembly ● Add 500uL Cold MeOH and 2 steel balls (3 mm diameter) and homogenize with GenoGrinder in two 30 sec intervals at 1500 rpm until a fine suspension is achieved. ● Centrifuge at 2C for 3 minutes spinning at 15,000rcf. Transfer methanol supernatant to 1.5 mL eppendorf vial containing 10uL 20% glycerol solution ● Add 500uL Cold MeOH and repeat homogenization with GenoGrinder in two 30 sec intervals at 1500 rpm until a fine suspension is achieved. Centrifuge again and add to the same 1.5mL tube ● Evaporated to dryness, or if a large number of samples has to be processed to a 96-well plate ● Re-suspend pellet in 100uL CUDA/PHAU 100nM MeOH:ACN ● Shake reconstitution for 1 minute at speed of 7 and let stand on wet ice for 15 minutes protected from light ● Shake reconstitution again and filter using PVDF Filter plate or Durapore Spin Filters. ● Transfer filtrate to glass insert in amber vial and cap or 96 well plate with pre-slit silicon mat ● Store at -20°C until LCMS analysis 5.3. Isolation of an enriched bile acid fraction from fecal water ● Label eppendorf tubes; add additional vials for blanks and controls ● Add 10uL Anti-Oxidant Solution using repeater pipetter or syringe repeater assembly ● Add 10uL SSTD using repeater pipetter or syringe repeater assembly ● Thaw out samples of fecal water, vortex briefly to homogenize, keep on ice ● Pipette 10uL of fecal water sample into labeled vials. ● Add 800uL of cold extraction solvent, acetonitrile containing 5% of ammonia ● Vortex samples, then shake for 6 minutes at 4 degree C ● Centrifuge for 3 minutes ● Transfer supernatant to fresh 1.5 mL eppendorf vials and evaporated to dryness, or for a large number of samples, to a 96-well plate and discard centrifugation residues ● Add 100µL CUDA/PHAU IS 100nM to dried sample, cap and vortex 10 sec to dissolve residues ● Set rack of samples on wet ice for 15min ● Filter using PVDF Plate or individual Spin Filters ● Transfer filtrates to glass insert in amber vial and cap or to 96 Well Plate ● Store at -20°C until LCMS analysis

Combined analysis:

Analysis ID	AN003300
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Vanquish
Column	Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type	ESI
MS instrument type	Ion trap
MS instrument name	ABI Sciex 6500+
Ion Mode	NEGATIVE
Units	pg/mg wet stool

Chromatography:

Chromatography ID:	CH002439
Instrument Name:	Vanquish
Column Name:	Waters Acquity BEH C18 (100 x 2mm,1.7um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003070
Analysis ID:	AN003300
Instrument Name:	ABI Sciex 6500+
Instrument Type:	Ion trap
MS Type:	ESI
MS Comments:	Extracts were analyzed by liquid chromatography (Waters ACQUITY UPLC I-Class system) coupled to a Sciex 6500+ QTRAP hybrid, triple quadrupole linear ion trap mass spectrometer. 5 µL of each extract was injected. Scheduled multiple reaction monitoring (MRM) was performed with optimized collision energies, de-clustering potentials, and collision cell exit potentials for individual analyte. A LC-MRM targeted method was used to analyze both bile acids and steroids with positive and negative polarity switching. Oxylipins were analyzed in another LC-MRM method in negative ionization mode only. All analytes were quantified against 6-point calibration curves using internal standards. Turbo Spray Ion Source parameters are: curtain gas (CUR) 25 psi, nebulizer gas (GS1) 50 psi, turbo-gas (GS2) 50 psi, electrospray voltage −4.5 kV/+3 kV, and source temperature 525 ◦C. Nitrogen was used as the collision gas. Software Analyst 1.6.3 and MultiQuant 3.0.2 (AB Sciex) were used for data acquisition and quantification. MRM transitions for the analytes are provided in the supplementary Tables S8 and S9
Ion Mode:	NEGATIVE