Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers

Skeletal Muscle

Matthew N. McCall¹  Karen Fox-Talbot²  Arun H. Patil²  Avi Z. Rosenberg²  Marc K. Halushka²  Katherine M. Fomchenko²  Xiaoping Yang²  Tim O. Nieuwenhuis²  Rohan X. Verma²  Clarisse Lukban³  David A. Kass³  Chulan Kwon³  Suraj Kannan³  Brian L. Lin³ 

[1] Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY, USA;Department of Pathology, Johns Hopkins University School of Medicine, Ross Bldg. Rm 632B, 720 Rutland Avenue, 21205, Baltimore, MD, USA;Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA;

关键词: Single cell RNA-sequencing;    Skeletal muscle;    Twitch;    Fiber;   

DOI  :  10.1186/s13395-021-00269-2

来源: Springer

【 摘 要 】

BackgroundSkeletal 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.MethodsWe 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.ResultsQuality 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.ConclusionThis 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.

【 授权许可】

CC BY