Year: 2021
Type: article
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.
Source: Skeletal Muscle
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,490
Funders: American Heart Association, University of Rochester, Georgia Clinical and Translational Science Alliance, National Institutes of Health, National Heart, Lung, and Blood Institute, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Environmental Health Sciences, National Institute of Child Health and Human Development, National Center for Advancing Translational Sciences, Eunice Kennedy Shriver National Institute of Child Health and Human Development
Awards: G1165598027, Regulation of Cardiac Progenitor Maintenance, G263750424, G2802014078, G4411448324, xMD-miRNA-Seq estimates cell-specific miRNA expression, G5258278944, G625544220, G7662493321, Canonical Wnt-Dependent Instructive Role for the First Heart Field in Second Heart Field Specification, Profiling Intercalated Disc Components in Arrhythmogenic Cardiomyopathy, G8898488596, G9000001345, G9000041231, G9000134567, G9000208853, Environmental Agents as Modulators of Disease Processes
OpenAlex ID: W3162469232