小鼠单肌纤维的RNA序列分析

Darren M. Blackburn; Felicia  Lazure; Vahab D. Soleimani

doi:10.21769/BioProtoc.3525

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

Peer-reviewed

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 email

(*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: 6193

评审: Gal HaimovichRuth A. FranklinAnonymous reviewer(s)

PDF

Q&A

引用

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

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

基于磁激活细胞分选（MACS）的小鼠肾足细胞分离方法

Jeffrey W. Pippin [...] Stuart J. Shankland

2025年07月05日 1633 阅读

RACE-Nano-Seq：解析基因组特定位点的转录组多样性

Lu Tang [...] Philipp Kapranov

2025年07月05日 1587 阅读

基于RNA邻位标记的OINC-seq方法分析RNA定位

Megan C. Pockalny [...] J. Matthew Taliaferro

2025年08月05日 965 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)

Skeletal muscle (骨骼肌)

SMART-Seq (SMART-Seq)

Gene expression analysis (基因表达分析)

Transcriptomics (转录组)

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

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

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)
Pipettes (P20, P200)
Diamond pen (VWR, catalog number: 201-0392)
Microscope (Fisher Scientific, inverted microscope, equipped with transmitted light and a 4x objective)
Centrifuge (Thermo Scientific, Sorval Legend Micro 21R Microcentrifuge, catalog number: 75002447)
Thermocycler (Bio-Rad, C1000 Touch Thermal Cycler, catalog number: 1851148)
Mini-centrifuge (Mandel, Benchmark MyFuge Minicentrifuge, catalog number: BEN-C1008-R)
Gel imager (LI-COR, Odyssey FC Imaging System)
qPCR machine (Applied Biosystems, QuantStudio7 Flex)
Bioanalyzer (Agilent, BioAnalyzer 2100)
Cell incubator (Thermo Scientific, Forma Series II Water-Jacketed CO₂ Incubator, catalog number:3110)
Vortex mixer (Mandel)
Power supply (Bio-Rad, PowerPac, catalog number: 1645050)
Microplate reader (BioTek, synergy4)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

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

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.
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.