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MB Sample ID: SA196420

Local Sample ID:	SRC-3 WT ZT20 replicate2 pos
Subject ID:	SU002162
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

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Subject:

Subject ID:	SU002162
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
SRC-3 WT ZT20 replicate2 pos	SA196420	FL023962	ZT20	ZT_time
SRC-3 WT ZT20 replicate2 pos	SA196420	FL023962	WT	Genotype

Collection:

Collection ID:	CO002155
Collection Summary:	To further define the 12-hour clock lipidome, we used a temporal lipidomics-based approach to identify hepatic lipid species in the mouse whole-liver. To unbiasedly characterize the 12-hour cycling lipidomes regulated by SRC-3, we profiled the temporal characteristics of oscillating lipidomes from adult male SRC-3 WT and KO littermates at 12-16 weeks of age under the same ZT condition (12h:12h LD cycle). We then sampled two biological replicates of mouse liver tissues at a temporal resolution starting at ZT0 and proceeding every four hours for two complete 12-hour cycles to determine whether SRC-3 ablation perturbs the 12-hour clock lipidome.
Sample Type:	Liver

Treatment:

Treatment ID:	TR002174
Treatment Summary:	Mouse liver lipids were extracted using a modified Bligh-Dyer method. Briefly, 50 mg of crushed issue sample from mouse whole liver was used. A 2:2:2 volume ratio of water/methanol/dichloromethane was used for lipid extract at room temperature after spiking internal standards 17:0 LPC, 17:0PC, 17:0 PE, 17:0 PG, 17:0 ceramide, 17:0 SM, 17:0PS, 17:0PA, 17:0 TAG, 17:0MAG, DAG 16:0/18:1, CE 17:0. The organic layer was collected followed by a complete drying procedure under nitrogen.

Treatment ID:

TR002174

Treatment Summary:

Mouse liver lipids were extracted using a modified Bligh-Dyer method. Briefly, 50 mg of crushed issue sample from mouse whole liver was used. A 2:2:2 volume ratio of water/methanol/dichloromethane was used for lipid extract at room temperature after spiking internal standards 17:0 LPC, 17:0PC, 17:0 PE, 17:0 PG, 17:0 ceramide, 17:0 SM, 17:0PS, 17:0PA, 17:0 TAG, 17:0MAG, DAG 16:0/18:1, CE 17:0. The organic layer was collected followed by a complete drying procedure under nitrogen.

Sample Preparation:

Sampleprep ID:	SP002168
Sampleprep Summary:	Before MS analysis, the dried extract was resuspended in 100 ?L of Buffer B (10:5:85 Acetonitrile/water/Isopropyl alcohol) containing 10mM NH4OAc and subjected to LC/MS. The lipidome was separated using reverse-phase chromatography. High-performance Liquid Chromatography (LC) grade water, methanol, acetonitrile, dichloromethane, isopropanol from Fisher scientific were used per the manufacturer’s instructions. Mass spectrometry (MS) grade lipid standards from Avanti Polar Lipids (Alabaster, AL) and MS grade ammonium acetate from sigma Aldrich (St. Louis, MO) were used. For internal standards and quality controls, the lipid stock solution was prepared by weighing an exact amount of the lipid internal standards in Chloroform/Methanol/H2O, resulting in sample aliquots at concentration of 1mg/mL as stock solutions. The stock solutions were diluted to 100 pmol/?L by mixing an appropriate volume of the internal standards LPC 17:0/0:0, PG 17:0/17:0, PE 17:0/17:0, PC 17:0/17:0, TAG 17:0/17:0/17:0, SM 18:1/17:0, MAG 17:0, DAG 16:0/18:1, CE 17:0, ceramide d 18:1/17:0, PA 17:0, PI 17:0/20:4, and PS 17:0/17:0. To monitor instrument performance, 10 ?L of a dried matrix-free mixture of the internal standards was used, reconstituted in 100 ?L of buffer B (5% water, 85%Isopropanolol: 10%Acetonitrile in 10mM NH4OAc). To monitor the lipid extraction process, a standard pool representing the tissue sample aliquots was used.Shimadzu CTO-20A Nexera X2 UHPLC systems was used for data acquisition, equipped with a degasser, binary pump, thermostat-regulated auto sampler, and a column oven for chromatographic separation. For lipid separation, 5 uL of the lipid extract was injected to a 1.8 ?m particle 50 ? 2.1 mm Acquity HSS UPLC T3 column (Waters, Milford, MA). The column heater temperature was set at 55° C. For chromatographic elution, a linear gradient was used over a 20 min total run time, with 60% Solvent A (acetonitrile/water (40:60, v/v) with 10 mM ammonium acetate) and 40% Solvent B (acetonitrile/water/isopropanol (10:5:85 v/v) with 10 mM ammonium acetate) gradient in the first 10 minutes. The gradient was ramped in a linear fashion to 100% Solvent B for 7 minutes. Then the system was switched back to 60% Solvent B and 40% Solvent A for 3 minutes. A flow rate of 0.4 mL/min was used at an injection volume of 5?L. The column was equilibrated for 3 min and run at a flow rate of 0.4 mL/min for a total run time of 20 min. The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source (AB Sciex, Concord, Canada).

Sampleprep ID:

SP002168

Sampleprep Summary:

Before MS analysis, the dried extract was resuspended in 100 ?L of Buffer B (10:5:85 Acetonitrile/water/Isopropyl alcohol) containing 10mM NH4OAc and subjected to LC/MS. The lipidome was separated using reverse-phase chromatography. High-performance Liquid Chromatography (LC) grade water, methanol, acetonitrile, dichloromethane, isopropanol from Fisher scientific were used per the manufacturer’s instructions. Mass spectrometry (MS) grade lipid standards from Avanti Polar Lipids (Alabaster, AL) and MS grade ammonium acetate from sigma Aldrich (St. Louis, MO) were used. For internal standards and quality controls, the lipid stock solution was prepared by weighing an exact amount of the lipid internal standards in Chloroform/Methanol/H2O, resulting in sample aliquots at concentration of 1mg/mL as stock solutions. The stock solutions were diluted to 100 pmol/?L by mixing an appropriate volume of the internal standards LPC 17:0/0:0, PG 17:0/17:0, PE 17:0/17:0, PC 17:0/17:0, TAG 17:0/17:0/17:0, SM 18:1/17:0, MAG 17:0, DAG 16:0/18:1, CE 17:0, ceramide d 18:1/17:0, PA 17:0, PI 17:0/20:4, and PS 17:0/17:0. To monitor instrument performance, 10 ?L of a dried matrix-free mixture of the internal standards was used, reconstituted in 100 ?L of buffer B (5% water, 85%Isopropanolol: 10%Acetonitrile in 10mM NH4OAc). To monitor the lipid extraction process, a standard pool representing the tissue sample aliquots was used.Shimadzu CTO-20A Nexera X2 UHPLC systems was used for data acquisition, equipped with a degasser, binary pump, thermostat-regulated auto sampler, and a column oven for chromatographic separation. For lipid separation, 5 uL of the lipid extract was injected to a 1.8 ?m particle 50 ? 2.1 mm Acquity HSS UPLC T3 column (Waters, Milford, MA). The column heater temperature was set at 55° C. For chromatographic elution, a linear gradient was used over a 20 min total run time, with 60% Solvent A (acetonitrile/water (40:60, v/v) with 10 mM ammonium acetate) and 40% Solvent B (acetonitrile/water/isopropanol (10:5:85 v/v) with 10 mM ammonium acetate) gradient in the first 10 minutes. The gradient was ramped in a linear fashion to 100% Solvent B for 7 minutes. Then the system was switched back to 60% Solvent B and 40% Solvent A for 3 minutes. A flow rate of 0.4 mL/min was used at an injection volume of 5?L. The column was equilibrated for 3 min and run at a flow rate of 0.4 mL/min for a total run time of 20 min. The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source (AB Sciex, Concord, Canada).

Combined analysis:

Analysis ID	AN003391	AN003392
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Shimadzu CTO-20A Nexera X2 UHPLC systems	Shimadzu CTO-20A Nexera X2 UHPLC systems
Column	Waters Acquity BEH HSS T3 (50 x 2.1mm,1.8um)	Waters Acquity BEH HSS T3 (50 x 2.1mm,1.8um)
MS Type	ESI	ESI
MS instrument type	Triple TOF	Triple TOF
MS instrument name	ABI Sciex 5600+ TripleTOF	ABI Sciex 5600+ TripleTOF
Ion Mode	POSITIVE	NEGATIVE
Units	Normalized intensity	Normalized intensity

Chromatography:

Chromatography ID:	CH002507
Instrument Name:	Shimadzu CTO-20A Nexera X2 UHPLC systems
Column Name:	Waters Acquity BEH HSS T3 (50 x 2.1mm,1.8um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003158
Analysis ID:	AN003391
Instrument Name:	ABI Sciex 5600+ TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source (AB Sciex, Concord, Canada).
Ion Mode:	POSITIVE

MS ID:	MS003159
Analysis ID:	AN003392
Instrument Name:	ABI Sciex 5600+ TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source (AB Sciex, Concord, Canada).
Ion Mode:	NEGATIVE