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

Anton Gulko; Adam Essene; Dylan Matthew Belmont-rausch; Molly Veregge; Deepti Pant; Danielle Tenen; Benedicte Schultz Kapel; Margo P. Emont; Tune H. Pers; Evan D. Rosen; Linus T. Tsai

doi:10.1016/j.xpro.2024.102893

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

Anton Gulko, Adam Essene, Dylan Matthew Belmont-rausch, Molly Veregge, Deepti Pant, Danielle Tenen, Benedicte Schultz Kapel, Margo P. Emont, Tune H. Pers, Evan D. Rosen, Linus T. Tsai^*

^*Corresponding author for this work

Research output: Contribution to journal › Journal article › Research › peer-review

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Abstract

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 103 Chromium controller.
This approach can eliminate batch confounding, and significantly reduces poorquality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information content
and cost efficiency.

Original language	English
Article number	102893
Journal	STAR Protocols
Volume	5
Issue number	1
Number of pages	21
ISSN	2666-1667
DOIs	https://doi.org/10.1016/j.xpro.2024.102893
Publication status	Published - 2024

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Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. / Gulko, Anton; Essene, Adam; Belmont-rausch, Dylan Matthew; Veregge, Molly; Pant, Deepti; Tenen, Danielle; Kapel, Benedicte Schultz; Emont, Margo P.; Pers, Tune H.; Rosen, Evan D.; Tsai, Linus T.

In: STAR Protocols, Vol. 5, No. 1, 102893, 2024.

Research output: Contribution to journal › Journal article › Research › peer-review

Gulko, Anton ; Essene, Adam ; Belmont-rausch, Dylan Matthew ; Veregge, Molly ; Pant, Deepti ; Tenen, Danielle ; Kapel, Benedicte Schultz ; Emont, Margo P. ; Pers, Tune H. ; Rosen, Evan D. ; Tsai, Linus T. / Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. In: STAR Protocols. 2024 ; Vol. 5, No. 1.

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AU - Pant, Deepti

AU - Tenen, Danielle

AU - Kapel, Benedicte Schultz

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AU - Pers, Tune H.

AU - Rosen, Evan D.

AU - Tsai, Linus T.

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AB - 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 adiposetissue samples, integrating flow cytometry for quality control, counting, and precise nuclei pooling for direct loading onto the popular 103 Chromium controller.This approach can eliminate batch confounding, and significantly reduces poorquality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information contentand cost efficiency.

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