Open AccessRevealing nascent RNA processing dynamics with nano-COP
Author(s) -
Heather L. Drexler,
Karine Choquet,
Hope E. Merens,
Paul S. Tang,
Jared T. Simpson,
L. Stirling Churchman
Publication year - 2021
Publication title -
nature protocols
Language(s) - English
Resource type - Journals
eISSN - 1754-2189
pISSN - 1750-2799
DOI - 10.1038/s41596-020-00469-y
Subject(s) - rna , polyadenylation , intron , rna splicing , post transcriptional modification , biology , transcription (linguistics) , computational biology , genetics , riboswitch , microbiology and biotechnology , non coding rna , gene , philosophy , linguistics
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.