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Homo sapiens

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

PROVIDER: PRJNA311053 | 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

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

2016-04-07 | GSE80039 | GEO

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

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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]

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

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CLIP-Seq of ZCCHC14 binding sites in HAV infected cells

Project description:We analyzed RNAs bound to FLAG tagged ZCCHC14 protein in HAV infected 293T cells using enhanced crosslinking-immunoprecipitation (eCLIP) and next generation sequencing (NGS), in order to identify ZCCHC14 binding sites on cellular and HAV RNAs.

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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

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