Summary of Study ST001415

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000971. The data can be accessed directly via it's Project DOI: 10.21228/M85X14 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST001415
Study Title	Multi-omic profiling of primary mouse neutrophils reveals a pattern of sex and age-related functional regulation
Study Summary	Neutrophils are the most abundant white blood cells in humans and constitute one of the first lines of defense in the innate immune response. Neutrophils are extremely short-lived cells, which survive less than a day after reaching terminal differentiation. Thus, little is known about how organismal aging, rather than the daily cellular aging process, may impact neutrophil biology. In addition, accumulating evidence suggests that both immunity and organismal aging are extremely sex-dimorphic. Here, we describe a multi-omic resource of mouse primary bone marrow neutrophils from young and old female and male animals, at the transcriptomic, metabolomic and lipidomic levels. Importantly, we identify widespread age-related and sex-dimorphic regulation of ‘omics’ in neutrophils, specifically regulation of chromatin metabolism. We leverage machine-learning and identify candidate molecular drivers of age-related and sex-dimorphic transcriptional regulation of neutrophils. We leverage our resource to predict increased levels/release of neutrophil elastase in male mice. To date, this dataset represents the largest multi-omic resource for the study of neutrophils across biological sex and ages. This resource identifies molecular states linked to neutrophil characteristics linked to organismal age or sex, which could be leveraged to improve immune responses across individuals.
Institute	Stanford University
Last Name	Contrepois
First Name	Kevin
Address	300 Pasteur Dr
Email	kcontrep@stanford.edu
Phone	6506664538
Submit Date	2020-06-30
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2021-06-30
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR000971
Project DOI:	doi: 10.21228/M85X14
Project Title:	Untargeted metabolomics of primary mouse neutrophils
Project Summary:	Untargeted metabolomics of primary neutrophils from young and old, male and female mice
Institute:	Stanford University
Department:	Genetics
Last Name:	Contrepois
First Name:	Kevin
Address:	300 Pasteur Dr, ALWAY bldg M302, STANFORD, California, 94305, USA
Email:	kcontrep@stanford.edu
Phone:	6507239914

Subject:

Subject ID:	SU001489
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6J
Age Or Age Range:	4 months & 20 months
Gender:	Male and female
Animal Animal Supplier:	Jackson Laboratories
Animal Housing:	SPF animal facility at USC
Species Group:	Mammals

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sex	Age
SA116246	F_20m_4a	Female	Old
SA116247	F_20m_4b	Female	Old
SA116248	F_20m_3a	Female	Old
SA116249	F_20m_3b	Female	Old
SA116250	F_4m_2b	Female	Young
SA116251	F_4m_1a	Female	Young
SA116252	F_4m_1b	Female	Young
SA116253	F_4m_2a	Female	Young
SA116254	M_20m_4b	Male	Old
SA116255	M_20m_3b	Male	Old
SA116256	M_20m_3a	Male	Old
SA116257	M_20m_4a	Male	Old
SA116258	M_4m_1a	Male	Young
SA116259	M_4m_2a	Male	Young
SA116260	M_4m_2b	Male	Young
SA116261	M_4m_1b	Male	Young

Showing results 1 to 16 of 16

Showing results 1 to 16 of 16

Collection:

Collection ID:	CO001484
Collection Summary:	The hindlimb bones of each mouse were harvested and kept on ice in D-PBS (Corning) supplemented with 1% Penicillin/Streptomycin (Corning) until further processing. Muscle tissue was removed from the bones, and the bone marrow from cleaned bones was collected into clean tubes (Amend et al., 2016). Red blood cells from the marrow were removed using Red Blood Cell Lysis (Miltenyi Biotech #130-094-183), according to the manufacturer’s instructions, albeit with no vortexing step to avoid unscheduled neutrophil activation. Neutrophils were isolated from other bone marrow cells using magnetic-assisted cell sorting (Miltenyi Biotech kit #130-097-658). Viability and yield were assessed using trypan blue exclusion and an automated COUNTESS cell counter (Thermo-Fisher Scientific). Purified cells were pelleted at 300g and snap-frozen in liquid nitrogen until processing for RNA, lipid or metabolite isolation.
Sample Type:	Bone marrow

Treatment:

Treatment ID:	TR001504
Treatment Summary:	There was no treatment.

Sample Preparation:

Sampleprep ID:	SP001497
Sampleprep Summary:	Metabolites and lipids were extracted from neutrophil cell pellets and analyzed in a randomized order. Extraction was performed using a biphasic separation protocol with ice-cold methanol, methyl tert-butyl ether (MTBE) and water (Contrepois et al., 2018). Briefly, 300μL of methanol spiked-in with 54 deuterated internal standards provided with the Lipidyzer platform (SCIEX, cat #5040156, LPISTDKIT-101) was added to the cell pellet, samples were vigorously vortexed for 20 seconds and sonicated in a water bath 3 times for 30 seconds on ice. Lipids were solubilized by adding 1000μL of MTBE and incubated under agitation for 1h at 4°C. After addition of 250μL of ice-cold water, the samples were vortexed for 1 min and centrifuged at 14,000g for 5 min at 20°C. The upper phase containing the lipids was then collected and dried down under nitrogen. The dry lipid extracts were reconstituted with 300μL of 10 mM ammonium acetate in 9:1 methanol:toluene for analaysis. The lower phase containing metabolites was subjected to further protein precipitation by adding 4 times of ice-cold 1:1:1 isopropanol:acetonitrile:water spiked in with 17 labeled internal standards and incubating for 2 hours at -20°C. The supernatant was dried down to completion under nitrogen and re-suspended in 100μL of 1:1 MeOH:Water for analysis.

Sampleprep ID:

SP001497

Sampleprep Summary:

Metabolites and lipids were extracted from neutrophil cell pellets and analyzed in a randomized order. Extraction was performed using a biphasic separation protocol with ice-cold methanol, methyl tert-butyl ether (MTBE) and water (Contrepois et al., 2018). Briefly, 300μL of methanol spiked-in with 54 deuterated internal standards provided with the Lipidyzer platform (SCIEX, cat #5040156, LPISTDKIT-101) was added to the cell pellet, samples were vigorously vortexed for 20 seconds and sonicated in a water bath 3 times for 30 seconds on ice. Lipids were solubilized by adding 1000μL of MTBE and incubated under agitation for 1h at 4°C. After addition of 250μL of ice-cold water, the samples were vortexed for 1 min and centrifuged at 14,000g for 5 min at 20°C. The upper phase containing the lipids was then collected and dried down under nitrogen. The dry lipid extracts were reconstituted with 300μL of 10 mM ammonium acetate in 9:1 methanol:toluene for analaysis. The lower phase containing metabolites was subjected to further protein precipitation by adding 4 times of ice-cold 1:1:1 isopropanol:acetonitrile:water spiked in with 17 labeled internal standards and incubating for 2 hours at -20°C. The supernatant was dried down to completion under nitrogen and re-suspended in 100μL of 1:1 MeOH:Water for analysis.

Chromatography:

Chromatography ID:	CH001735
Chromatography Summary:	HILIC experiments were performed using a ZIC-HILIC column 2.1x100 mm, 3.5μm, 200Å (Merck Millipore) and mobile phase solvents consisting of 10mM ammonium acetate in 50/50 acetonitrile/water (A) and 10 mM ammonium acetate in 95/5 acetonitrile/water (B).(Contrepois et al., 2015)
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:	40
Flow Rate:	0.5 ml/min
Solvent A:	95% acetonitrile/5% water; 10 mM ammonium acetate
Solvent B:	95% acetonitrile/5% water; 10 mM ammonium acetate
Chromatography Type:	HILIC

Chromatography ID:	CH001736
Chromatography Summary:	RPLC experiments were performed using a Zorbax SBaq column 2.1 x 50 mm, 1.7 μm, 100Å (Agilent Technologies) and mobile phase solvents consisting of 0.06% acetic acid in water (A) and 0.06% acetic acid in methanol (B). (Contrepois et al., 2015)
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Agilent Zorbax SBaq (50 x 2.1mm,1.7um)
Column Temperature:	60
Flow Rate:	0.6 ml/min
Solvent A:	100% water; 0.06% acetic acid
Solvent B:	100% methanol; 0.06% acetic acid
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN002365
Analysis Type:	MS
Operator Name:	Kevin Contrepois
Detector Type:	Orbitrap
Data Format:	.RAW
Chromatography ID:	CH001735
Num Factors:	4
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST001415_AN002365_Results.txt
Units:	MS count

Analysis ID:	AN002366
Analysis Type:	MS
Operator Name:	Kevin Contrepois
Detector Type:	Orbitrap
Data Format:	.RAW
Chromatography ID:	CH001735
Num Factors:	4
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST001415_AN002366_Results.txt
Units:	MS count

Analysis ID:	AN002367
Analysis Type:	MS
Operator Name:	Kevin Contrepois
Detector Type:	Orbitrap
Data Format:	.RAW
Chromatography ID:	CH001736
Num Factors:	4
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST001415_AN002367_Results.txt
Units:	MS count

Analysis ID:	AN002368
Analysis Type:	MS
Operator Name:	Kevin Contrepois
Detector Type:	Orbitrap
Data Format:	.RAW
Chromatography ID:	CH001736
Num Factors:	4
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST001415_AN002368_Results.txt
Units:	MS count