核糖核酸
聚腺苷酸
内含子
RNA剪接
转录后修饰
生物
抄写(语言学)
计算生物学
遗传学
核糖开关
细胞生物学
非编码RNA
基因
语言学
哲学
作者
Heather L. Drexler,Karine Choquet,Hope E. Merens,Paul S. Tang,Jared T. Simpson,L. Stirling Churchman
出处
期刊:Nature Protocols
[Springer Nature]
日期:2021-01-29
卷期号:16 (3): 1343-1375
被引量:27
标识
DOI:10.1038/s41596-020-00469-y
摘要
During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3'-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3' ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read-based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.
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