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Enhanced CLIP sequencing (eCLIP & Ribo-eCLIP)

What is eCLIP?

As RNA binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNAs, 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.1,2,3

 eCLIP workflow: from preparation of crosslinked cells and lysates, to purification of RBP-RNA, to library preparation and bioinformatic analysis (peak calling)

1. Van Nostrand, Eric L., et al. "Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)." Nature Methods 13.6 (2016): 508-514.2.
2. Van Nostrand, Eric L., et al. "Robust, cost-effective profiling of RNA binding protein targets with single-end enhanced crosslinking and immunoprecipitation (seCLIP)." mRNA Processing: Methods and Protocols (2017): 177-200.3.
3. Blue, Steven M., et al. "Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq." Nature Protocols 17.5 (2022): 1223-1265.

Applications

eCLIP is a powerful technique used to map RNA-binding protein (RBP) binding sites across the entire transcriptome. This technique has a wide range of applications that provide valuable insights into the regulation of target transcripts by RBPs. Below are some key applications of eCLIP:

Maps RBP binding sites and identifies RNA targets: eCLIP maps the binding sites of RBP on RNA molecules. Mapping the binding sites to specific sites within the transcript (5’UTR, splice sites, CDS etc.) gives insight into how an RBP regulates its target transcripts. Additionally,  the identity of these target transcripts also informs what biological functions the RBP regulates. 

Functional Annotation of Non-Coding RNAs: eCLIP can be used to study the binding of RBPs to non-coding RNAs, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). This sheds light on the regulatory networks involving these non-coding RNAs.

Disease Mechanisms: Researchers can use eCLIP to investigate how dysregulation of RBPs and their binding to RNA contribute to various diseases, including cancer, neurodegenerative disorders, and autoimmune conditions. 

Drug Discovery and Development: Identifying RBP binding sites can aid in drug discovery by uncovering novel targets for small molecule drugs or RNA-based therapeutics. It can also be used to study the effects of RNA-targeted drugs on RBP-RNA interactions.

Comparative Analysis: eCLIP data can be compared across different cell types, tissues, or conditions to gain a comprehensive understanding of how RBP binding patterns vary in different biological contexts.

Functional Genomics: The integration of eCLIP datasets such as transcriptomics, RNA half-life, differential splicing, and proteomics can help pinpoint the function RBP has on its RNA targets.

Translation Regulation: Ribo-eCLIP data maps the ribosome-associated RNAs and the data can be used to analyze translation efficiency.

Project Workflow

Project Initiation: 

  • Contact the RNA Center at RNAcenter@ucsd.edu
  • Discuss experimental designs and goals
  • Discuss cost estimates, expectations, and potential outcomes

Sample Preparation:

  • Prepare samples according to protocol for either cell lines, primary cells, or tissues.
  • Antibody validation needed, either use validated antibody (ENCODE) or core will charge to validate new antibody.
  • At least one test sample is recommended for new antibody or cell type.

Sample Submission:

  • Submit Sample Manifest according to instructions.
  • Drop off samples or ship samples.

Sample Processing:

  • The RNA Center will validate antibody according to ENCODE standards.
  • Total RNA levels will be calculated before starting the eCLIP protocol.
  • Validation (CT values, library size QC).
  • Libraries sent for sequencing.

Data Transfer:

  • Generate and transfer data.
  • If agreed upon, data analysis and processing.

Materials and Resources

Download Sample Submission Guide (Excel)

Download ENCODE crosslinking guide (Word file)

Download ENCODE Antibody Validation Protocol (Word file)

Download Sample Submission Manifest (Word file)