Preparation of Mammalian Nascent RNA for Long Read Sequencing.
Preparation of Mammalian Nascent RNA for Long Read Sequencing.
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DOI：
10.1002/cpmb.128
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发表时间：
2020-12
影响因子：
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通讯作者：
Neugebauer KM
中科院分区：
文献类型：
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作者：
Reimer KA;Neugebauer KM
长读测序技术现在可以对数百到数千个核苷酸长的RNA（或它们的cdna）进行高质量的测序。新生RNA的长读序列提供了共转录RNA加工事件的单核苷酸分辨率信息，如剪接、折叠和碱基修饰。在这里，我们描述了如何通过染色质相关RNA的亚细胞分离从哺乳动物细胞中分离新生RNA，以及如何耗尽polyA+ RNA和rRNA，最后，如何生成全长cDNA文库用于长读测序平台。这种方法通过揭示剪接模式或其他传统短读RNA测序无法获得的RNA加工事件，可以了解跨多内含子转录本的协调剪接状态。
Long read sequencing technologies now allow high-quality sequencing of RNA (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single-nucleotide resolution information about co-transcriptional RNA processing events – e.g. splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin-associated RNA, as well as how to deplete polyA+ RNA and rRNA, and finally, how to generate a full-length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi-intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing.