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

Abstract

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).

Funds to V.S. were provided by the NIAMS-IRP through NIH grants AR041126 and AR041164. Work in P.M.-C’s laboratory was supported by MINECO-RTI2018-096068, ERC-AdG-741966, LaCaixa-HEALTH-HR17-00040, UPGRADE-H2020-825825 and Marató-TV3, MDM-2014-0370, and SEV-2015-0505. Authors acknowledge technical support from the UPF/CRG and CNIC flow cytometry units.