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Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative

Authors :
Elisabeth F. Heuston
Ayo P. Doumatey
Faiza Naz
Shamima Islam
Stacie Anderson
Martha R. Kirby
Stephen Wincovitch
Stefania Dell’Orso
Charles N. Rotimi
Adebowale A. Adeyemo
Source :
Communications Biology, Vol 8, Iss 1, Pp 1-9 (2025)
Publication Year :
2025
Publisher :
Nature Portfolio, 2025.

Abstract

Abstract Single cell studies have transformed our understanding of cellular heterogeneity in disease but the need for fresh starting material can be an obstacle, especially in the context of international multicenter studies and archived tissue. We developed a protocol to obtain high-quality cells and nuclei from dissected human skeletal muscle archived in the preservative Allprotect® Tissue Reagent. After fluorescent imaging microscopy confirmed intact nuclei, we performed four protocol variations that compared sequencing metrics between cells and nuclei enriched by either filtering or flow cytometry sorting. Cells and nuclei (either sorted or filtered) produced statistically identical transcriptional profiles and recapitulated 8 cell types present in skeletal muscle. Flow cytometry sorting successfully enriched for higher-quality cells and nuclei but resulted in an overall decrease in input material. Our protocol provides an important resource for obtaining high-quality single cell genomic material from archived tissue and to streamline global collaborative efforts.

Subjects

Subjects :
Biology (General)
QH301-705.5

Details

Language :
English
ISSN :
23993642
Volume :
8
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Communications Biology
Publication Type :
Academic Journal
Accession number :
edsdoj.1e2aa4eba1b1448aa7a7ace4667ea9a9
Document Type :
article
Full Text :
https://doi.org/10.1038/s42003-024-07445-2