Genomic Technology Section, NIAMS, NIH, Bethesda, MD 20892, USA.
Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, NIH, Bethesda, MD 20892, USA.
STAR Protoc. 2021 Apr 15;2(2):100451. doi: 10.1016/j.xpro.2021.100451. eCollection 2021 Jun 18.
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).
从稀有细胞群体的 RNA 中生成可重复、高质量的 cDNA 文库仍然具有挑战性。在这里，我们描述了一种高通量 RNA-seq 文库制备的方案，包括通过荧光激活细胞分选从小鼠胫骨前肌中分离 200 个骨骼肌干细胞和 cDNA 制备。我们还描述了从分化的小鼠胚胎干细胞中提取 RNA 和制备 cDNA。有关该方案使用和执行的完整详细信息，请参阅 Juan 等人（2016 年）和 Garcia-Prat 等人（2016 年）。
