title: Single-nucleus RNA-seq of differentiating human myoblasts reveals the extent of fate heterogeneity.
abstract: Myoblasts are precursor skeletal muscle cells that differentiate into fused, multinucleated myotubes. Current single-cell microfluidic methods are not optimized for capturing very large, multinucleated cells such as myotubes. To circumvent the problem, we performed single-nucleus transcriptome analysis. Using immortalized human myoblasts, we performed RNA-seq analysis of single cells (scRNA-seq) and single nuclei (snRNA-seq) and found them comparable, with a distinct enrichment for long non-coding RNAs (lncRNAs) in snRNA-seq. We then compared snRNA-seq of myoblasts before and after differentiation. We observed the presence of mononucleated cells (MNCs) that remained unfused and analyzed separately from multi-nucleated myotubes. We found that while the transcriptome profiles of myoblast and myotube nuclei are relatively homogeneous, MNC nuclei exhibited significant heterogeneity, with the majority of them adopting a distinct mesenchymal state. Primary transcripts for microRNAs (miRNAs) that participate in skeletal muscle differentiation were among the most differentially expressed lncRNAs, which we validated using NanoString. Our study demonstrates that snRNA-seq provides reliable transcriptome quantification for cells that are otherwise not amenable to current single-cell platforms. Our results further indicate that snRNA-seq has unique advantage in capturing nucleus-enriched lncRNAs and miRNA precursors that are useful in mapping and monitoring differential miRNA expression during cellular differentiation.
authors: Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, USA.|Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697-2280, USA.
authors: Weihua
authors: W
authors: Zeng
authors: Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, USA.|Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697-2280, USA.
authors: Shan
authors: S
authors: Jiang
authors: Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA 92697-1700, USA.
authors: Xiangduo
authors: X
authors: Kong
authors: Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, USA.|Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697-2280, USA.
authors: Nicole
authors: N
authors: El-Ali
authors: Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA 92697-1700, USA.
authors: Alexander R
authors: AR
authors: Ball
authors: Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA 92697-1700, USA.
authors: Christopher I-Hsing
authors: CI
authors: Ma
authors: Department of Regenerative Medicine, National Center for Geriatrics and Gerontology, 7-430 Morioka, Oobu, Aichi 474-8522, Japan.
authors: Naohiro
authors: N
authors: Hashimoto
authors: Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA 92697-1700, USA kyokomor@uci.edu.
authors: Kyoko
authors: K
authors: Yokomori
authors: Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, USA ali.mortazavi@uci.edu.|Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697-2280, USA.
authors: Ali
authors: A
authors: Mortazavi
mesh_terms: D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology; D035683:MicroRNAs / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D032446:Myoblasts / Q000166:cytology* / Q000502:physiology; D051787:Myogenic Regulatory Factor 5 / Q000235:genetics; D062085:RNA, Long Noncoding; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods
chemical_list: D035683:MicroRNAs; D051787:Myogenic Regulatory Factor 5; D062085:RNA, Long Noncoding; C525710:MIR222, human; C497417:MYF5 protein, human