Molecular Genetics Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, USA.
Section on Molecular and Cell Biology, NICHD, NIH, Bethesda, MD, USA.
Methods Mol Biol. 2024;2723:285-301. doi: 10.1007/978-1-0716-3481-3_17.
The polyadenylation of the 3' ends of messenger RNAs is an important regulator of stability and translation. We developed the single-molecule poly(A) tail sequencing method, SM-PATseq, to assay tail lengths of the whole transcriptome at nucleotide resolution using long-read sequencing. This method generates cDNA using an oligo-dT 3' splint adaptor ligation to prime first-strand cDNA synthesis, followed by random hexamer priming for second-strand synthesis. By directly sequencing the cDNA on long-read platforms, we can resolve tail lengths at nucleotide resolution, identify non-A bases within the tail, and quantify transcript abundance analogous to traditional RNAseq methods. Here, we discuss the method for generating, sequencing, and primary analysis of poly(A) tail data from total RNA using the Pacific Biosciences Sequel platform.
信使 RNA 3' 端的多聚腺苷酸化是稳定性和翻译的重要调节剂。我们开发了单分子多(A)尾测序方法 SM-PATseq，使用长读测序技术在核苷酸分辨率下测定整个转录组的尾巴长度。该方法使用 oligo-dT 3' 分裂接头连接生成 cDNA，然后使用随机六聚体启动子进行第二链合成。通过在长读平台上直接测序 cDNA，我们可以在核苷酸分辨率下解析尾巴长度，识别尾巴内的非 A 碱基，并定量类似于传统 RNAseq 方法的转录本丰度。在这里，我们讨论了使用 Pacific Biosciences Sequel 平台从总 RNA 生成、测序和初步分析多(A)尾数据的方法。
