Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative
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 a...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-01-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-024-07445-2 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Elisabeth F. Heuston |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-1e2aa4eba1b1448aa7a7ace4667ea9a9 |
| institution | Kabale University |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-1e2aa4eba1b1448aa7a7ace4667ea9a92025-01-05T12:43:11ZengNature PortfolioCommunications Biology2399-36422025-01-01811910.1038/s42003-024-07445-2Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservativeElisabeth F. Heuston0Ayo P. Doumatey1Faiza Naz2Shamima Islam3Stacie Anderson4Martha R. Kirby5Stephen Wincovitch6Stefania Dell’Orso7Charles N. Rotimi8Adebowale A. Adeyemo9Center for Research on Genomics and Global Health, National Human Genome Research Institute, National Institutes of HealthCenter for Research on Genomics and Global Health, National Human Genome Research Institute, National Institutes of HealthGenomic Technology Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of HealthGenomic Technology Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of HealthNHGRI Flow Cytometry Core, National Human Genome Research Institute, National Institutes of HealthNHGRI Flow Cytometry Core, National Human Genome Research Institute, National Institutes of HealthAdvanced Imaging & Analysis Core, National Human Genome Research Institute, NIHGenomic Technology Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of HealthCenter for Research on Genomics and Global Health, National Human Genome Research Institute, National Institutes of HealthCenter for Research on Genomics and Global Health, National Human Genome Research Institute, National Institutes of HealthAbstract 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.https://doi.org/10.1038/s42003-024-07445-2 |
| spellingShingle | 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 Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative Communications Biology |
| title | Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative |
| title_full | Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative |
| title_fullStr | Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative |
| title_full_unstemmed | Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative |
| title_short | Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative |
| title_sort | optimized methods for scrna seq and snrna seq of skeletal muscle stored in nucleic acid stabilizing preservative |
| url | https://doi.org/10.1038/s42003-024-07445-2 |
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