RIP-Seq
RNA Immunoprecipitation Sequencing
RIP-Seq maps the sites at which proteins are bound to the RNA within RNA-protein complexes (Zhao et al., 2010). In this method, RNA-protein complexes are immunoprecipitated with antibodies targeted to the protein of interest. After RNase digestion, RNA protected by protein binding is extracted and reverse-transcribed to cDNA. The locations can then be mapped back to the genome. Deep sequencing of cDNA provides single-base resolution of protein-bound RNA.
Advantages:
- Maps specific protein-RNA complexes, such as polycomb-associated RNAs
- Low background and higher resolution of binding site due to RNase digestion
- No prior knowledge of the RNA is required
- Genome-wide RNA screen
Disadvantages:
- Requires antibodies to the targeted proteins
- Nonspecific antibodies will precipitate nonspecific complexes
- Lack of crosslinking or stabilization of the complexes may lead to false negatives
- RNase digestion must be controlled carefully
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
McFadden E. J. and Hargrove A. E. Biochemical Methods To Investigate lncRNA and the Influence of lncRNA:Protein Complexes on Chromatin. Biochemistry. 2016;55:1615-1630
Fang Y. and Fullwood M. J. Roles, Functions, and Mechanisms of Long Non-coding RNAs in Cancer. Genomics Proteomics Bioinformatics. 2016;14:42-54
Kim T. K. and Shiekhattar R. Diverse regulatory interactions of long noncoding RNAs. Curr Opin Genet Dev. 2016;36:73-82
Goeman F. and Fanciulli M. Application of RNA-Seq Technology in Cancer Chemoprevention. Cancer Chemoprevention: Methods and Protocols. 2016;31-43
Carrier M. C., Lalaouna D. and Masse E. A game of tag: MAPS catches up on RNA interactomes. RNA Biol. 2016;13:473-476
References:
Cloonan S. M., Glass K., Laucho-Contreras M. E., et al. Mitochondrial iron chelation ameliorates cigarette smoke-induced bronchitis and emphysema in mice. Nat Med. 2016;22:163-174
Yabe-Wada T., Matsuba S., Takeda K., et al. TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin. Sci Rep. 2016;6:26566
Dugar G., Svensson S. L., Bischler T., et al. The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Nat Commun. 2016;7:11667
Ennajdaoui H., Howard J. M., Sterne-Weiler T., et al. IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC. Cell Rep. 2016;15:1876-1883
Faraji F., Hu Y., Yang H. H., et al. Post-transcriptional Control of Tumor Cell Autonomous Metastatic Potential by CCR4-NOT Deadenylase CNOT7. PLoS Genet. 2016;12:e1005820
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History: RIP-Seq
Revision by sbrumpton on 2017-06-21 07:50:25 - Show/Hide
RNA Immunoprecipitation Sequencing
RIP-Seq maps the sites at which proteins are bound to the RNA within RNA-protein complexes (Zhao et al., 2010). In this method, RNA-protein complexes are immunoprecipitated with antibodies targeted to the protein of interest. After RNase digestion, RNA protected by protein binding is extracted and reverse-transcribed to cDNA. The locations can then be mapped back to the genome. Deep sequencing of cDNA provides single-base resolution of protein-bound RNA.
Advantages:- Maps specific protein-RNA complexes, such as polycomb-associated RNAs
- Low background and higher resolution of binding site due to RNase digestion
- No prior knowledge of the RNA is required
- Genome-wide RNA screen
Disadvantages:- Requires antibodies to the targeted proteins
- Nonspecific antibodies will precipitate nonspecific complexes
- Lack of crosslinking or stabilization of the complexes may lead to false negatives
- RNase digestion must be controlled carefully
Reagents:Illumina Library prep and Array Kit SelectorReviews:McFadden E. J. and Hargrove A. E. Biochemical Methods To Investigate lncRNA and the Influence of lncRNA:Protein Complexes on Chromatin. Biochemistry. 2016;55:1615-1630Fang Y. and Fullwood M. J. Roles, Functions, and Mechanisms of Long Non-coding RNAs in Cancer. Genomics Proteomics Bioinformatics. 2016;14:42-54Kim T. K. and Shiekhattar R. Diverse regulatory interactions of long noncoding RNAs. Curr Opin Genet Dev. 2016;36:73-82Goeman F. and Fanciulli M. Application of RNA-Seq Technology in Cancer Chemoprevention. Cancer Chemoprevention: Methods and Protocols. 2016;31-43Carrier M. C., Lalaouna D. and Masse E. A game of tag: MAPS catches up on RNA interactomes. RNA Biol. 2016;13:473-476References:Cloonan S. M., Glass K., Laucho-Contreras M. E., et al. Mitochondrial iron chelation ameliorates cigarette smoke-induced bronchitis and emphysema in mice. Nat Med. 2016;22:163-174Yabe-Wada T., Matsuba S., Takeda K., et al. TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin. Sci Rep. 2016;6:26566Dugar G., Svensson S. L., Bischler T., et al. The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Nat Commun. 2016;7:11667Ennajdaoui H., Howard J. M., Sterne-Weiler T., et al. IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC. Cell Rep. 2016;15:1876-1883Faraji F., Hu Y., Yang H. H., et al. Post-transcriptional Control of Tumor Cell Autonomous Metastatic Potential by CCR4-NOT Deadenylase CNOT7. PLoS Genet. 2016;12:e1005820