Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC, USA.
Integrative Bioinformatics, Biostatistics and Computational Biology Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC, USA.
Methods Mol Biol. 2024;2723:253-266. doi: 10.1007/978-1-0716-3481-3_15.
Poly(A) tail metabolism is critical for various biological processes, including early embryogenesis and cell differentiation. While traditional biochemical methods to measure poly(A) tail length allow for the study of selected transcripts, the advent of long-read sequencing technologies enabled the development of simple and robust protocols to measure poly(A) tail length at the transcriptome level. Here, we describe a direct RNA sequencing protocol to capture poly(A) tail terminal additions based on the splint ligation of barcoded oligos compatible with terminal guanylation and uridylation. We cover how to prepare the libraries and perform the bioinformatics analysis to simultaneously determine the length of the transcripts' poly(A) tails and detect the presence of terminal guanylation and uridylation.
聚腺苷酸化尾代谢对于各种生物学过程至关重要，包括早期胚胎发生和细胞分化。虽然传统的生化方法可以测量聚腺苷酸化尾的长度，从而研究选定的转录本，但长读测序技术的出现使得开发在转录组水平上测量聚腺苷酸化尾长度的简单而稳健的方法成为可能。在这里，我们描述了一种基于带有末端鸟苷酸化和尿苷酸化的条形码寡核苷酸的衔接子连接，直接从 RNA 中捕获聚腺苷酸化尾末端添加的 RNA 测序方法。我们介绍了如何准备文库并进行生物信息学分析，以同时确定转录物的聚腺苷酸化尾的长度，并检测末端鸟苷酸化和尿苷酸化的存在。
