Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
STAR Protoc. 2021 Aug 25;2(3):100651. doi: 10.1016/j.xpro.2021.100651. eCollection 2021 Sep 17.
The protocol allows for labeling nascent RNA without isolating nuclei. The cell-permeable uridine analog, 5-ethynyluridine (EU), is added to media to allow labeling of nascent transcripts. Cells are lysed, total RNA is collected, and biotin is conjugated to EU-labeled RNAs. Custom biotin RNAs are added and biotinylated RNAs are isolated for generation of cDNA libraries. The sequencing data are normalized to controls for quantitative assessment of the nascent transcriptome. The protocol takes 4 days, not including sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Palozola et al. (2017).
该方案允许在不分离细胞核的情况下对新生 RNA 进行标记。将细胞可渗透的尿嘧啶类似物 5-乙炔基尿嘧啶（EU）添加到培养基中，以允许对新生转录本进行标记。细胞裂解，收集总 RNA，并将生物素连接到 EU 标记的 RNA 上。添加定制的生物素 RNA，并分离生物素化 RNA，用于生成 cDNA 文库。测序数据被标准化为对照，以定量评估新生转录组。该方案需要 4 天时间，不包括测序和分析。有关该方案使用和执行的完整详细信息，请参见 Palozola 等人（2017 年）。
