Revealing nascent RNA processing dynamics with nano-COP.
Nat Protoc
; 16(3): 1343-1375, 2021 03.
Article
em En
| MEDLINE
| ID: mdl-33514943
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Precursores de RNA
/
Análise de Sequência de RNA
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Modificação Traducional de Proteínas
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article