CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins.
Methods
; 118-119: 50-59, 2017 04 15.
Article
em En
| MEDLINE
| ID: mdl-28003131
ABSTRACT
Identification of in vivo direct RNA targets for RNA binding proteins (RBPs) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus. Further, we show that TAG-eCLIP performed using tag-specific antibodies can yield the same robust binding profiles after proper control normalization as eCLIP with antibodies against endogenous proteins. Finally, we note that antibodies against commonly used tags can immunoprecipitate significant amounts of antibody-specific RNA, emphasizing the need for paired controls alongside each experiment for normalization. TAG-eCLIP enables eCLIP profiling of new native proteins where no suitable antibody exists, expanding the RBP-RNA interaction landscape.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Coloração e Rotulagem
/
RNA
/
Biblioteca Gênica
/
Proteínas de Ligação a RNA
/
Sequenciamento de Nucleotídeos em Larga Escala
/
Sistemas CRISPR-Cas
Limite:
Humans
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article