Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing.

Cell isolation Cell separation/fractionation Flow Cytometry Genomics Molecular Biology RNA-seq Sequencing Single Cell

Journal

STAR protocols
ISSN: 2666-1667
Titre abrégé: STAR Protoc
Pays: United States
ID NLM: 101769501

Informations de publication

Date de publication:
15 Mar 2024
Historique:
received: 15 10 2023
revised: 27 12 2023
accepted: 30 01 2024
pubmed: 28 2 2024
medline: 28 2 2024
entrez: 28 2 2024
Statut: ppublish

Résumé

Adipocyte size and fragility and commercial kit costs impose significant limitations on single-cell RNA sequencing of adipose tissue. Accordingly, we developed a workflow to isolate and sample-barcode nuclei from individual adipose tissue samples, integrating flow cytometry for quality control, counting, and precise nuclei pooling for direct loading onto the popular 10× Chromium controller. This approach can eliminate batch confounding, and significantly reduces poor-quality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information content and cost efficiency.

Identifiants

DOI: 10.1016/j.xpro.2024.102893 PMID: 38416649 PMC: PMC10909897
pubmed: 38416649
pii: S2666-1667(24)00058-3
doi: 10.1016/j.xpro.2024.102893
pmc: PMC10909897
pii:
doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

102893

Subventions

Organisme : NIDDK NIH HHS
ID : K01 DK134806
Pays : United States
Organisme : NIDDK NIH HHS
ID : P30 DK046200
Pays : United States
Organisme : NIDDK NIH HHS
ID : RC2 DK116691
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK128406
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK135541
Pays : United States
Organisme : NHLBI NIH HHS
ID : P01 HL149630
Pays : United States
Organisme : NIDDK NIH HHS
ID : P30 DK135043
Pays : United States

Informations de copyright

Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of interests The authors declare no competing interests.

Auteurs

Anton Gulko (A)

Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; Boston Nutrition and Obesity Research Center/Boston Area Diabetes and Endocrinology Research Center Functional Genomics and Bioinformatics Core, Boston, MA, USA.

Adam Essene (A)

Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; Boston Nutrition and Obesity Research Center/Boston Area Diabetes and Endocrinology Research Center Functional Genomics and Bioinformatics Core, Boston, MA, USA.

Dylan Matthew Belmont-Rausch (DM)

Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, 2200 Copenhagen, Denmark.

Molly Veregge (M)

Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; Boston Nutrition and Obesity Research Center/Boston Area Diabetes and Endocrinology Research Center Functional Genomics and Bioinformatics Core, Boston, MA, USA.

Deepti Pant (D)

Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; Boston Nutrition and Obesity Research Center/Boston Area Diabetes and Endocrinology Research Center Functional Genomics and Bioinformatics Core, Boston, MA, USA.

Danielle Tenen (D)

Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; Boston Nutrition and Obesity Research Center/Boston Area Diabetes and Endocrinology Research Center Functional Genomics and Bioinformatics Core, Boston, MA, USA.

Benedicte Schultz Kapel (BS)

Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, 2200 Copenhagen, Denmark.

Margo P Emont (MP)

Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA.

Tune H Pers (TH)

Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, 2200 Copenhagen, Denmark.

Evan D Rosen (ED)

Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; Boston Nutrition and Obesity Research Center/Boston Area Diabetes and Endocrinology Research Center Functional Genomics and Bioinformatics Core, Boston, MA, USA.

Linus T Tsai (LT)

Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; Boston Nutrition and Obesity Research Center/Boston Area Diabetes and Endocrinology Research Center Functional Genomics and Bioinformatics Core, Boston, MA, USA. Electronic address: ltsai@bidmc.harvard.edu.

Classifications MeSH