Transcriptome-wide measurement of poly(A) tail length and composition at subnanogram total RNA sensitivity by PAIso-seq
Transcriptome-wide measurement of poly(A) tail length and composition at subnanogram total RNA sensitivity by PAIso-seq
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DOI：
10.1038/s41596-022-00704-8
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发表时间：
2022-07-13
期刊：
影响因子：
14.8
通讯作者：
Lu, Falong
中科院分区：
文献类型：
--
作者：
Liu, Yusheng;Zhang, Yiwei;Lu, Falong
Poly(A) 尾以非模板化方式添加到大多数 mRNA 的 3' 末端，在转录后调节（包括 mRNA 输出、稳定性和翻译）中发挥重要作用。测量多聚 (A) 尾对于了解其在生物和医学研究几乎各个方面的调节作用至关重要。以前分析poly(A)尾的方法需要大量的输入RNA（微克级总RNA），这限制了它们的应用。我们最近开发了一种基于 PacBio 测序的单卵母细胞水平灵敏度（单个哺乳动物卵母细胞含有约 0.5 ng 总 RNA）的 Poly(A) 包含全长 RNA 同工型测序方法 (PAlso-seq)，该方法能够准确测量 Poly(A) 尾长度和 Poly(A) 尾体内的非 A 残基以及全长 cDNA，从而提供了使用非常有限的输入材料研究珍贵的体内样品的机会。在这里，我们描述了从单个小鼠卵母细胞或大量卵母细胞样本中制备 PAlso-seq 文库的详细方案。此外，我们提供完整的生物信息学管道来执行从原始数据到下游分析的分析。所需的最短时间类似于 PAlso-seq 双链 cDNA 制备 14.5 小时，HiFi 模式下 PacBio 测序 2 d，初始数据分析 8 小时。
Poly(A) tails are added to the 3' ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Measuring poly(A) tails is critical for understanding their regulatory roles in almost every aspect of biological and medical studies. Previous methods for analyzing poly(A) tails require large amounts of input RNA (microgram-level total RNA), which limits their application. We recently developed a poly(A) inclusive full-length RNA isoform-sequencing method (PAlso-seq) at single-oocyte-level sensitivity (a single mammalian oocyte contains similar to 0.5 ng of total RNA) based on PacBio sequencing that enabled accurate measurement of the poly(A) tail length and non-A residues within the body of poly(A) tails along with the full-length cDNA, providing the opportunity to study precious in vivo samples with very limited input material. Here, we describe a detailed protocol for PAlso-seq library preparation from single mouse oocytes or bulk oocyte samples. In addition, we provide a complete bioinformatic pipeline to perform the analysis from the raw data to downstream analysis. The minimum time required is similar to 14.5 h for PAlso-seq double-stranded cDNA preparation, 2 d for PacBio sequencing in HiFi mode and 8 h for the initial data analysis.