CLIP-Seq or HITS-CLIP
High-Throughput Sequencing of CLIP cDNA Library
![](/wp-content/plugins/presserly-wiki/images/CLIP-Seq or HITS-CLIP.png)
Crosslinking and immunoprecipitation (CLIP), with the use of RNase T1 trimming, was first described by Ule et al (Ule et al., 2005). and later applied to high-throughput sequencing to map protein-RNA binding sites in vivo (Licatalosi et al., 2008) (Chi et al., 2009). This approach is similar to RIP-Seq but uses crosslinking to stabilize the protein-RNA complexes.
In HITS-CLIP, RNA-protein complexes are UV-crosslinked and immunoprecipitated. The protein-RNA complexes are treated with RNase T1, followed by proteinase K. RNA is extracted and reverse-transcribed to cDNA. Deep sequencing of the cDNA provides single-base resolution mapping of protein binding sites on RNA.
An improvement on the HITS-CLIP protocol was published by Gillen et al., which reduces artifacts from mispriming occurences (Gillen et al., 2016).
Other versions: iCLIP, irCLIP, eCLIP, miCLIP
Advantages:
- Crosslinking stabilizes the protein-target binding
- UV crosslinking can be carried out in vivo
- Provides 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:
- Over-representation of the RT complement due to mispriming (Gillen et al., 2016)
- Antibodies not specific to the target may precipitate nonspecific complexes.
- UV crosslinking is not efficient and requires close protein-RNA interactions
- Artifacts may be introduced during the crosslinking process
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Cook K. B., Hughes T. R. and Morris Q. D. High-throughput characterization of protein-RNA interactions. Brief Funct Genomics. 2015;14:74-89
References:
Van Haute L., Dietmann S., Kremer L., et al. Deficient methylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying mutations in NSUN3. Nat Commun. 2016;7:12039
Vourekas A., Alexiou P., Vrettos N., Maragkakis M. and Mourelatos Z. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature. 2016;531:390-394
Ji Z., Song R., Huang H., Regev A. and Struhl K. Transcriptome-scale RNase-footprinting of RNA-protein complexes. Nat Biotechnol. 2016;34:410-413
Gosline S. J., Gurtan A. M., JnBaptiste C. K., et al. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements. Cell Rep. 2016;14:310-319
Nutter C. A., Jaworski E. A., Verma S. K., et al. Dysregulation of RBFOX2 Is an Early Event in Cardiac Pathogenesis of Diabetes. Cell Rep. 2016;15:2200-2213
Spengler R. M., Zhang X., Cheng C., et al. Elucidation of transcriptome-wide microRNA binding sites in human cardiac tissues by Ago2 HITS-CLIP. Nucleic Acids Res. 2016;
Gillen A. E., Yamamoto T. M., Kline E., Hesselberth J. R. and Kabos P. Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts. BMC Genomics. 2016;17:338
Moore M. J., Zhang C., Gantman E. C., Mele A., Darnell J. C. and Darnell R. B. Erratum: Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis. Nat Protoc. 2016;11:616
Bennett C. G., Riemondy K., Chapnick D. A., Bunker E., Liu X., et al. Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration. Nucleic Acids Res. 2016;44:3788-3800
Vourekas A., Alexiou P., Vrettos N., Maragkakis M. and Mourelatos Z. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature. 2016;531:390-394
Zheng Q., Bao C., Guo W., et al. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat Commun. 2016;7:11215
Preusse M., Theis F. J. and Mueller N. S. miTALOS v2: Analyzing Tissue Specific microRNA Function. PLoS One. 2016;11:e0151771
Kapeli K., Pratt G. A., Vu A. Q., et al. Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses. Nat Commun. 2016;7:12143
Valentin-Vega Y. A., Wang Y. D., Parker M., et al. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation. Sci Rep. 2016;6:25996
Chen S., Blank M. F., Iyer A., et al. SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing. Nat Commun. 2016;7:10734
Zhao H., Chen M., Lind S. B. and Pettersson U. Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection. Virology. 2016;492:242-250
Taliaferro J. M., Vidaki M., Oliveira R., et al. Distal Alternative Last Exons Localize mRNAs to Neural Projections. Mol Cell. 2016;61:821-833
Gaudreau M. C., Grapton D., Helness A., et al. Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells. Sci Rep. 2016;6:27379
Dugar G., Svensson S. L., Bischler T., Waldchen S., Reinhardt R., et al. The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Nat Commun. 2016;7:11667
Related
History: CLIP-Seq or HITS-CLIP
Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide
High-Throughput Sequencing of CLIP cDNA Library
![](/wp-content/plugins/presserly-wiki/images/CLIP-Seq or HITS-CLIP.png)
Crosslinking and immunoprecipitation (CLIP), with the use of RNase T1 trimming, was first described by Ule et al (Ule et al., 2005). and later applied to high-throughput sequencing to map protein-RNA binding sites in vivo (Licatalosi et al., 2008) (Chi et al., 2009). This approach is similar to RIP-Seq but uses crosslinking to stabilize the protein-RNA complexes.
In HITS-CLIP, RNA-protein complexes are UV-crosslinked and immunoprecipitated. The protein-RNA complexes are treated with RNase T1, followed by proteinase K. RNA is extracted and reverse-transcribed to cDNA. Deep sequencing of the cDNA provides single-base resolution mapping of protein binding sites on RNA.
An improvement on the HITS-CLIP protocol was published by Gillen et al., which reduces artifacts from mispriming occurences (Gillen et al., 2016).
Other versions: iCLIP, irCLIP, eCLIP, miCLIP
Advantages:- Crosslinking stabilizes the protein-target binding
- UV crosslinking can be carried out in vivo
- Provides 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:- Over-representation of the RT complement due to mispriming (Gillen et al., 2016)
- Antibodies not specific to the target may precipitate nonspecific complexes.
- UV crosslinking is not efficient and requires close protein-RNA interactions
- Artifacts may be introduced during the crosslinking process
Reagents:Illumina Library prep and Array Kit SelectorReviews:Cook K. B., Hughes T. R. and Morris Q. D. High-throughput characterization of protein-RNA interactions. Brief Funct Genomics. 2015;14:74-89References:Van Haute L., Dietmann S., Kremer L., et al. Deficient methylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying mutations in NSUN3. Nat Commun. 2016;7:12039Vourekas A., Alexiou P., Vrettos N., Maragkakis M. and Mourelatos Z. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature. 2016;531:390-394Ji Z., Song R., Huang H., Regev A. and Struhl K. Transcriptome-scale RNase-footprinting of RNA-protein complexes. Nat Biotechnol. 2016;34:410-413Gosline S. J., Gurtan A. M., JnBaptiste C. K., et al. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements. Cell Rep. 2016;14:310-319Nutter C. A., Jaworski E. A., Verma S. K., et al. Dysregulation of RBFOX2 Is an Early Event in Cardiac Pathogenesis of Diabetes. Cell Rep. 2016;15:2200-2213Spengler R. M., Zhang X., Cheng C., et al. Elucidation of transcriptome-wide microRNA binding sites in human cardiac tissues by Ago2 HITS-CLIP. Nucleic Acids Res. 2016;Gillen A. E., Yamamoto T. M., Kline E., Hesselberth J. R. and Kabos P. Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts. BMC Genomics. 2016;17:338Moore M. J., Zhang C., Gantman E. C., Mele A., Darnell J. C. and Darnell R. B. Erratum: Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis. Nat Protoc. 2016;11:616Bennett C. G., Riemondy K., Chapnick D. A., Bunker E., Liu X., et al. Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration. Nucleic Acids Res. 2016;44:3788-3800Vourekas A., Alexiou P., Vrettos N., Maragkakis M. and Mourelatos Z. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature. 2016;531:390-394Zheng Q., Bao C., Guo W., et al. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat Commun. 2016;7:11215Preusse M., Theis F. J. and Mueller N. S. miTALOS v2: Analyzing Tissue Specific microRNA Function. PLoS One. 2016;11:e0151771Kapeli K., Pratt G. A., Vu A. Q., et al. Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses. Nat Commun. 2016;7:12143Valentin-Vega Y. A., Wang Y. D., Parker M., et al. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation. Sci Rep. 2016;6:25996Chen S., Blank M. F., Iyer A., et al. SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing. Nat Commun. 2016;7:10734Zhao H., Chen M., Lind S. B. and Pettersson U. Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection. Virology. 2016;492:242-250Taliaferro J. M., Vidaki M., Oliveira R., et al. Distal Alternative Last Exons Localize mRNAs to Neural Projections. Mol Cell. 2016;61:821-833Gaudreau M. C., Grapton D., Helness A., et al. Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells. Sci Rep. 2016;6:27379Dugar G., Svensson S. L., Bischler T., Waldchen S., Reinhardt R., et al. The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Nat Commun. 2016;7:11667