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Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites

ABSTRACT: Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites

PROVIDER: PRJNA311057 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR3147600_1.fastq.gz	Fastqsanger.gz
	SRR3147600_2.fastq.gz	Fastqsanger.gz
	SRR3147610_1.fastq.gz	Fastqsanger.gz
	SRR3147610_2.fastq.gz	Fastqsanger.gz
	SRR3147620_1.fastq.gz	Fastqsanger.gz

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Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites [eCLIP - 293T cells]

Project description:RNA binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNAs. As defects in protein-RNA recognition lead to human disease, UV-crosslinking and immunoprecipitation (CLIP) of ribonuclear complexes followed by deep sequencing (-seq) is critical in constructing protein-RNA maps to expand our understanding of RBP function. However, current CLIP protocols are technically demanding and involve low complexity libraries that yield squandered sequencing of PCR duplicates and high experimental failure rates. To enable truly large-scale implementation of CLIP-seq, we have developed an enhanced CLIP methodology (eCLIP) that features a decrease of ~10 cycles of requisite amplification with a concomitant >60% decrease in discarded PCR duplicate reads, while maintaining the ability to identify RNA binding with single-nucleotide resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP also dramatically improves specificity in discovery of authentic binding sites. To demonstrate that eCLIP enables large-scale and robust profiling of RBPs, 102 eCLIP experiments in biological duplicate for a diverse collection of 74 RBPs in HepG2 and K562 cells were completed (available at https://www.encodeproject.org). We establish that eCLIP is comparable in amplification and sample requirements to ChIP-seq, and enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP experiments and RNA-centric perspectives of RBP activity.

2016-03-28 | GSE77629 | GEO

Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites [eCLIP - HepG2/K562 cells]

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Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites [iCLIP]

2016-03-28 | GSE77633 | GEO

Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites [array]

2016-03-28 | GSE77339 | GEO

Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).

Project description:As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity.

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Multiplexed transcriptome discovery of RNA binding protein binding sites by antibody-barcode eCLIP

Project description:UV cross-linking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the current enhanced CLIP (eCLIP) protocol still requires ~4 days of hands-on time and lacks the ability to scale. We present a new method termed antibody barcode CLIP (ABC) that utilizes DNA-barcoded antibodies to multiplex CLIP detection methods. We demonstrate the scalability and simplicity of ABC by performing CLIP on multiple RBPs simultaneously, minimizing sample-to-sample variation, and maintaining the same material requirement for a single eCLIP experiment.

2022-10-18 | GSE205536 | GEO

Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2016-03-28 | E-GEOD-77634 | biostudies-arrayexpress

CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins

Project description: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.

2017-08-22 | GSE88722 | GEO

Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [eCLIP-Seq]

Project description:Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using a recently developed enhanced UV crosslinking and immunoprecipitation (eCLIP) approach, we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3'UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. eCLIP-seq was performed in biological replicate for IGF2BP1/IMP1 and IGF2BP2/IMP2, as well as one replicate each for IGF2BP3/IMP3, RBFOX2, and an IgG control. Each sample has a size-matched input control for analysis

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Enhanced CLIP (eCLIP) identified differential UPF1 binding sites in mouse neural progenitor cells (NPCs) samples

Project description:Enhanced CLIP (eCLIP) identified differential UPF1 binding sites in mouse neural progenitor cells (NPCs) samples

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