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| Status |
Public on Aug 22, 2017 |
| Title |
CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Identification of in vivo direct RNA targets for RNA binding proteins (RBP) 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-seq) 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 landscape.
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| |
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| Overall design |
eCLIP-seq performed in wild-type and endogenously tagged HEK293T cell lines
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| |
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| Contributor(s) |
Van Nostrand E, Gelboin-Burkhart C, Wang R, Pratt GA, Blue S, Yeo G |
| Citation(s) |
28003131 |
| |
| Submission date |
Oct 13, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (43)
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| Relations |
| BioProject |
PRJNA348371 |
| SRA |
SRP091517 |
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