Capturing 5′ and 3′ native ends of mRNAs concurrently with Akron sequencing
Capturing 5′ and 3′ native ends of mRNAs concurrently with Akron sequencing
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DOI：
10.1038/s41596-019-0151-9
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发表时间：
2019-05-01
期刊：
影响因子：
14.8
通讯作者：
Mourelatos, Zissimos
中科院分区：
文献类型：
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作者：
Ibrahim, Fadia;Mourelatos, Zissimos
RNA测序方法的进展已经揭示了RNA代谢的许多方面，但仅限于调查RNA的3'或5'末端，因此缺失了如果捕获两端则可以揭示的机制方面。我们开发了Akron测序（Akron-seq），这是一种从相同的输入RNA平行捕获未加帽的多腺苷酸化mRNA的天然5'末端和加帽mRNA的3'末端的方法。因此，Akron-seq独特地能够以单核苷酸分辨率评估全长和截短的mRNA。Akron-seq涉及RNA分离，核糖体和丰富的小加帽RNA的消耗，以及加帽和聚腺苷酸化mRNA的选择。mRNA的内源性末端通过衔接子连接标记，然后进行片段化、cDNA生成、PCR扩增和深度测序。我们在这里描述的逐步方案是针对培养的人类细胞进行优化的，但也可以适用于原代细胞和组织。Akron-seq可在6天内完成，测序和分析可在6天内完成。
Advances in RNA-sequencing methods have uncovered many aspects of RNA metabolism but are limited to surveying either the 3' or 5' terminus of RNAs, thus missing mechanistic aspects that could be revealed if both ends were captured. We developed Akron sequencing (Akron-seq), a method that captures in parallel the native 5' ends of uncapped, polyadenylated mRNAs and 3' ends of capped mRNAs from the same input RNA. Thus, Akron-seq uniquely enables assessment of full-length and truncated mRNAs at single-nucleotide resolution. Akron-seq involves RNA isolation, depletion of ribosomal and abundant small capped RNAs, and selection of capped and polyadenylated mRNAs. The endogenous ends of mRNAs are marked by adaptor ligation, followed by fragmentation, cDNA generation, PCR amplification, and deep sequencing. The step-by-step protocol we describe here is optimized for cultured human cells but can be adapted to primary cells and tissues. Akron-seq can be completed within 6 d, and sequencing and analysis can be completed within 6 d.