Department of Physiology, Guangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.
Stanley Center for Psychiatric Research, The Broad Institute, Cambridge, MA, USA.
Methods Mol Biol. 2022;2537:51-62. doi: 10.1007/978-1-0716-2521-7_4.
Alternative splicing of pre-mRNAs expands the coding abilities of genomes by generating distinct transcription variants from individual genes. It contributes to the marvelous complexity of the transcriptome in neurons. Given the differential expression of alternative splicing regulators and diversity in alternative splicing programs in neuronal subpopulations, it is urgent and necessary to develop methods to efficiently isolate diverse subgroups of neurons and analyze their transcriptomic diversity. Here, we describe a protocol to isolate RNA from specific neuronal types using a fluorescence-activated cell sorting (FACS)-based method to analyze alternative splicing events in a cell type-specific manner. The method is universally applicable to analyze alternative splicing in fluorescent protein-labeled neuronal types. It was optimized to preserve the transcription state and improve efficiency in cell suspension purification. With our protocol, fluorescent protein-labeled neurons could be efficiently purified. The transcription states suitable for gene expression and alternative splicing analysis could be well-preserved.
前体 mRNA 的可变剪接通过从单个基因产生不同的转录变体，从而扩展基因组的编码能力。它有助于神经元中转录组的惊人复杂性。鉴于可变剪接调节因子的差异表达和神经元亚群中可变剪接程序的多样性，迫切需要开发方法来有效地分离不同的神经元亚群并分析它们的转录组多样性。在这里，我们描述了一种使用基于荧光激活细胞分选（FACS）的方法从特定神经元类型中分离 RNA 的方案，以便以细胞类型特异性的方式分析可变剪接事件。该方法普遍适用于分析荧光蛋白标记的神经元类型中的可变剪接。它经过优化，可保留转录状态并提高细胞悬浮液纯化的效率。通过我们的方案，可以有效地纯化荧光蛋白标记的神经元。适合基因表达和可变剪接分析的转录状态可以得到很好的保留。
