Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

RNA Sequencing of Single Myofibers from Mus musculus

小鼠单肌纤维的RNA序列分析

DB Darren M. Blackburn *
FL Felicia Lazure *
VS Vahab D. Soleimani

 (*contributed equally to this work) 发布: 2020年02月20日第10卷第4期 DOI: 10.21769/BioProtoc.3525 浏览次数: 7277

评审: Gal HaimovichRuth A. FranklinAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Biological Chemistry, featuring study using the protocol.

Dec 2019

Advertisement

“科研可重复性与创新”系列网络研讨会
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

基于Langendorff灌流的小鼠心肌细胞简化分离方法

基于Langendorff灌流的小鼠心肌细胞简化分离方法

Mie S. Larsen [...] Tamzin Zawadzki

2026年07月20日 223 阅读

逆转录病毒制备后卫星细胞的分离培养与感染

逆转录病毒制备后卫星细胞的分离培养与感染

Chuanli Zhou [...] Elizabeth H. Chen

2026年07月20日 154 阅读

利用流式细胞术分离单核与双核肝细胞

利用流式细胞术分离单核与双核肝细胞

Yusuke V. Watanabe [...] Takeshi Katsuda

2026年07月20日 199 阅读

Abstract

Whole transcriptome analysis is a key method in biology that allows researchers to determine the effect a condition has on gene regulation. One difficulty in RNA sequencing of muscle is that traditional methods are performed on the whole muscle, but this captures non-myogenic cells that are part of the muscle. In order to analyze only the transcriptome of myofibers we combine single myofiber isolation with SMART-Seq to provide high resolution genome wide expression of a single myofiber.

Keywords: Single myofiber (单肌纤维) search Skeletal muscle (骨骼肌) search SMART-Seq (SMART-Seq) search Gene expression analysis (基因表达分析) search Transcriptomics (转录组) search

Background

Differences in gene expression are key indicators used in studies to see the effect of disease and ageing on cell and tissue types. Switching Mechanism at 5′ end of RNA Templates Sequencing (SMART-Seq) is a powerful technique that enables high resolution sequencing of mRNA from a limited number of cells (Picelli et al., 2014).

Traditionally, RNA sequencing of muscle has been performed on the whole muscle (Pette et al., 1999). However, muscle is a very heterogeneous tissue that is composed of a variety of cell types, such as adipocytes, endothelial cells, mesenchymal cells, and fibroblasts (Arnold et al., 2007; Christov et al., 2007; Joe et al., 2010; Giordani et al., 2019). Adding to this problem is the variety of different types of fibers, with two main groups known as the fast and slow twitch fibers (Brooke and Kaiser, 1970; Pette and Staron, 1997). In order to clearly determine the changes in transcriptome that arise solely from the muscle fiber without the confounding presence of the other cell types, the fiber must be isolated from these cell types.

Here, we combine single myofiber isolation with SMART-Seq to perform a high-resolution sequencing of mRNA at a quality comparable to traditional whole muscle sequencing, while removing the presence of the other non-myogenic cell types. This method will allow researchers to analyze changes in the transcriptome in myofibers under different conditions, such as disease and ageing, and to distinguish the transcriptome of slow and fast fiber types. We successfully implemented this method to analyze the differences in the whole transcriptome of single myofibers isolated from young and old mice.

Materials and Reagents

  1. Materials
    1. Pipette tips
    2. 6-well plate (TC-tested) (Sarstedt, catalog number: 83.3920)
    3. Glass Pasteur pipettes (VWR, catalog number: 14672-200)
    4. 1.5 ml Microtubes (Sarstedt, catalog number: 72.690.300)
    5. 0.2 ml Microtubes (Sarstedt, catalog number: 72.737)
    6. Magnetic rack (Thermo Fisher, DynaMag-2 Magnet, catalog number: 12321D)
    7. 0.22 micron filter (UltiDent, catalog number: 229747)
    8. Large bore glass pipette
    9. Small bore glass pipette

  2. Animal
    C57BL/6 mouse (Procedure has been successfully performed on mice aged from 1 month to 24 months of age)

  3. Reagents
    For digestion of the extensor digitorum longus
    1. PBS (Wisent, catalog number: 311-425-CL)
    2. Collagenase from Clostridium histolyticum (Sigma, catalog number: C0130-1G)
    3. DMEM (Invitrogen, catalog number: 11995073)
    4. Horse Serum (Wisent, catalog number: 065-250)
    5. Trypsin (Gibco, catalog number: 15090-046)
    6. Isoflurane (Fresenius Kabi, catalog number: CP0406V2)
    7. CO2 (Praxair, catalog number: GP-700500)
    8. Digestion buffer (see Recipes)
    9. Coating media (see Recipes)

    For extraction of RNA from a single myofiber
    1. RNase free water
    2. RNase inhibitors (from SMART-Seq HT kit, Takara BIO USA, catalog number: 634456)
    3. SMART-Seq Reaction Buffer (see Recipes)

    For cDNA library preparation
    1. SMART-Seq HT kit (Takara BIO USA, catalog number: 634456)

    For library purification
    1. Ampure XP beads (Beckman Coulter Life Sciences, catalog number: A63882)
    2. Ethanol (Commercial Alcohols, catalog number: P016EAAN)
    3. RNase free water

    For preparing sequencing ready libraries
    1. Nextera XT DNA Library Preparation kit (Illumina, catalog number: FC-131-1024)
    2. Nextera XT Index kit (Illumina, catalog number: FC-131-1001)
    3. Quant-IT Picogreen dsDNA Assay kit (Invitrogen, catalog number: P7589)

    For purification and size selection
    1. Ampure XP beads (Beckman Coulter Life Sciences, catalog number: A63882)
    2. Ethanol (Commercial Alcohols, catalog number: P016EAAN)
    3. RNase free water
    4. Quant-IT Picogreen dsDNA Assay kit (Invitrogen, catalog number: P7589)

    For quality control
    1. Agarose (Bioshop, catalog number: AGA001.500)
    2. Tris (Hydroxymethyl) Amino Methane (Multicell, catalog number: 600-125-LG)
    3. Glacial acetic acid (Fisherbrand, catalog number: 351272-212)
    4. Na2EDTA·2H2O (Bioshop, catalog number: EDT 001)
    5. GelGreen Nucleic Acid Gel Stain (Biotium, catalog number: 41005)
    6. SYBR Green (Applied Biosystems, catalog number: A25742)
    7. Primers (Integrated DNA Technologies, standard desalting, 25nmole) (Table 1)
    8. 100 bp DNA ladder (GenedireX, catalog number: DM001-R500)
    9. TAE Buffer (see Recipes)

      Table 1. Primer sequences used for quality control of libraries

Equipment

  1. Dissection tools
    1. Fine point high precision forceps (Fisher Scientific, catalog number: 22-327379)
    2. Sharp-Pointed dissecting scissors (Fisher Scientific, catalog number: 08-935)
  2. Pipettes (P20, P200)
  3. Diamond pen (VWR, catalog number: 201-0392)
  4. Microscope (Fisher Scientific, inverted microscope, equipped with transmitted light and a 4x objective)
  5. Centrifuge (Thermo Scientific, Sorval Legend Micro 21R Microcentrifuge, catalog number: 75002447)
  6. Thermocycler (Bio-Rad, C1000 Touch Thermal Cycler, catalog number: 1851148)
  7. Mini-centrifuge (Mandel, Benchmark MyFuge Minicentrifuge, catalog number: BEN-C1008-R)
  8. Gel imager (LI-COR, Odyssey FC Imaging System)
  9. qPCR machine (Applied Biosystems, QuantStudio7 Flex)
  10. Bioanalyzer (Agilent, BioAnalyzer 2100)
  11. Cell incubator (Thermo Scientific, Forma Series II Water-Jacketed CO2 Incubator, catalog number:3110)
  12. Vortex mixer (Mandel)
  13. Power supply (Bio-Rad, PowerPac, catalog number: 1645050)
  14. Microplate reader (BioTek, synergy4)

Procedure

English
中文翻译

文章信息

版权信息

© 2020 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Blackburn, D. M., Lazure, F. and Soleimani, V. D. (2020). RNA Sequencing of Single Myofibers from Mus musculus. Bio-protocol 10(4): e3525. DOI: 10.21769/BioProtoc.3525.
  2. Blackburn, D. M., Lazure, F., Corchado, A. H., Perkins, T. J., Najafabadi, H. S. and Soleimani, V. D. (2019). High-resolution genome-wide expression analysis of single myofibers using SMART-Seq. J Biol Chem 294(52): 20097-20108.

    3. ris ris Download Citation in RIS Format

分类

发育生物学 > 细胞生长和命运决定 > 肌纤维

分子生物学 > RNA > RNA 测序

细胞生物学 > 细胞分离和培养 > 细胞分离

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件