Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers
Abstract Background Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorti...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-05-01
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| Series: | Skeletal Muscle |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13395-021-00269-2 |
| _version_ | 1818939929141444608 |
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| author | Rohan X. Verma Suraj Kannan Brian L. Lin Katherine M. Fomchenko Tim O. Nieuwenhuis Arun H. Patil Clarisse Lukban Xiaoping Yang Karen Fox-Talbot Matthew N. McCall Chulan Kwon David A. Kass Avi Z. Rosenberg Marc K. Halushka |
| author_facet | Rohan X. Verma Suraj Kannan Brian L. Lin Katherine M. Fomchenko Tim O. Nieuwenhuis Arun H. Patil Clarisse Lukban Xiaoping Yang Karen Fox-Talbot Matthew N. McCall Chulan Kwon David A. Kass Avi Z. Rosenberg Marc K. Halushka |
| author_sort | Rohan X. Verma |
| collection | DOAJ |
| description | Abstract Background Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type. Methods We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers. Results Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types. Conclusion This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes. |
| first_indexed | 2024-12-20T06:31:33Z |
| format | Article |
| id | doaj.art-4c8d78ac599048469743702ada0190f6 |
| institution | Directory Open Access Journal |
| issn | 2044-5040 |
| language | English |
| last_indexed | 2024-12-20T06:31:33Z |
| publishDate | 2021-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Skeletal Muscle |
| spelling | doaj.art-4c8d78ac599048469743702ada0190f62022-12-21T19:50:07ZengBMCSkeletal Muscle2044-50402021-05-0111111010.1186/s13395-021-00269-2Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibersRohan X. Verma0Suraj Kannan1Brian L. Lin2Katherine M. Fomchenko3Tim O. Nieuwenhuis4Arun H. Patil5Clarisse Lukban6Xiaoping Yang7Karen Fox-Talbot8Matthew N. McCall9Chulan Kwon10David A. Kass11Avi Z. Rosenberg12Marc K. Halushka13Department of Pathology, Johns Hopkins University School of MedicineDivision of Cardiology, Department of Medicine, Johns Hopkins University School of MedicineDivision of Cardiology, Department of Medicine, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDivision of Cardiology, Department of Medicine, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Biostatistics and Computational Biology, University of Rochester Medical CenterDivision of Cardiology, Department of Medicine, Johns Hopkins University School of MedicineDivision of Cardiology, Department of Medicine, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineAbstract Background Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type. Methods We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers. Results Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types. Conclusion This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.https://doi.org/10.1186/s13395-021-00269-2Single cell RNA-sequencingSkeletal muscleTwitchFiber |
| spellingShingle | Rohan X. Verma Suraj Kannan Brian L. Lin Katherine M. Fomchenko Tim O. Nieuwenhuis Arun H. Patil Clarisse Lukban Xiaoping Yang Karen Fox-Talbot Matthew N. McCall Chulan Kwon David A. Kass Avi Z. Rosenberg Marc K. Halushka Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers Skeletal Muscle Single cell RNA-sequencing Skeletal muscle Twitch Fiber |
| title | Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers |
| title_full | Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers |
| title_fullStr | Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers |
| title_full_unstemmed | Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers |
| title_short | Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers |
| title_sort | single cell rna seq analysis of the flexor digitorum brevis mouse myofibers |
| topic | Single cell RNA-sequencing Skeletal muscle Twitch Fiber |
| url | https://doi.org/10.1186/s13395-021-00269-2 |
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