Published April 15, 2021 | Version v1
Journal article Open

Protocol for RNA-seq library preparation starting from a rare muscle stem cell population or a limited number of mouse embryonic stem cells

  • 1. Genomic Technology Section, NIAMS, NIH, Bethesda, MD 20892, USA
  • 2. Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, NIH, Bethesda, MD 20892, USA
  • 3. Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED) and ICREA, Barcelona, Spain - Spanish National Center on Cardiovascular Research (CNIC), Madrid, Spain
  • 4. Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED) and ICREA, Barcelona, Spain - Spanish National Center on Cardiovascular Research (CNIC), Madrid, Spain - Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada

Description

Summary

It remains challenging to generate reproducible, high-quality cDNA libraries from RNA derived from rare cell populations. Here, we describe a protocol for high-throughput RNA-seq library preparation, including isolation of 200 skeletal muscle stem cells from mouse tibialis anterior muscle by fluorescence-activated cell sorting and cDNA preparation. We also describe RNA extraction and cDNA preparation from differentiating mouse embryonic stem cells.

For complete details on the use and execution of this protocol, please refer to Juan et al. (2016) and Garcia-Prat et al. (2016).

 

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Funding

UPGRADE – Unlocking Precision Gene Therapy 825825
European Commission
STEM-AGING – Tissue regeneration and aging: the decisive quiescent stem-cell state 741966
European Commission