CLIP-Seq Service

Background

What is CLIP-Seq?

CLIP-Seq (Purple Immunoprecipitation Binding High-throughput sequencing) is a new method to reveal the relationships between RNA molecules and RNA-binding proteins (RBPs) at the genome scale. The key concept behind it is the use of ultraviolet light irradiation to make RNA and the protein that it binds to covalently bond and cross-link. This stabilizes the RNA-protein complex so that target RBPs and RNA stretches they bind can be immunoprecipitated to identify them. Then, these RNA segments were spliced, reverse transcribed and PCR amplified, and finally high-throughput sequenced. RNA-binding sites and RBP patterns can be predicted by bioinformatics to better understand the function of RBPs after transcription and how they affect life.

Fig. 1. Schematic overview of the core steps common to most variants of the cross-linking and immunoprecipitation (CLIP) protocol. (Hafner, et al., 2021)

Upgrade of CLIP-Seq

Since the CLIP technique was first proposed in 2003, it has undergone several important upgrades to improve the accuracy and efficiency of RNA-protein interaction analysis:

• iCLIP (2010): iCLIP technology enables single-base resolution RNA-protein interaction analysis through cDNA cyclization, significantly improving the accuracy of RNA binding sites.
• eCLIP (2015): eCLIP, as an enhanced version of CLIP, improves the RBPs target recognition rate by 2-3 times and optimizes the RNA library preparation procedure by adding 3'RNA splice and 3' single-stranded DNA splice to immunoprecipitated magnetic beads.
• hiCLIP (2017): The hiCLIP technique combines the benefits of iCLIP and eCLIP to identify specific RNA complexes that bind to RBP at the full transcriptome level. One of its characteristics is the ability to capture two strands of RNA that the RBP binds to at the same time and join the two ends of the double-stranded RNA by ligase.

Applications of CLIP-Seq

There are numerous applications for the CLIP-Seq method (see below:
Map RNA-RBP Co-Relations: CLIP-Seq allows to describe interactions between Argonaute proteins, miRNAs and mRNAs which is important for gene expression regulation.
Identifying RBP and RNA Networks: This method allows us to identify what and how RNA-binding proteins interact with RNA-coding RNAs such as miRNAs and lncRNAs.
Direct RBP-RNA Interaction Detection: CLIP-Seq, unlike RIP-seq, detects direct RBP-RNA interactions and will provide exact binding sites for RNAs such as miRNA and lncRNA.
miRNA Targets: In miRNA targets detection, CLIP-Seq minimizes false positive predictions and simplifies the search for miRNA binding sites.
Functional Research with lncRNA/circRNA: it’s used to study how lncRNAs and circRNAs operate.
RBP Mechanism Exploration: The method helps identify the action mechanism of RNA-binding proteins, which is critical for studying RNA’s biological activity.
m6A Modification Mapping: miCLIP-seq is one version that maps m6A modifications throughout the transcriptome at single-base resolution.
Post-transcriptional Control: CLIP-Seq participates in RNA splicing, transport, sequence editing, intracellular localization, and translation regulation.

Service Procedure

Workflow

Platforms

Our CLIP-Seq services leverage state-of-the-art methods and sophisticated equipment. Utilizing UV cross-linking and RNA-IP, paired with high-throughput sequencers, enables us to achieve exceptional sensitivity and data depth. With tools like cross-linkers, fluorescent quantitative PCR machines, and automated sample prep systems, alongside high-end sequencing platforms, we ensure precision and efficiency. Our technology excels in accurately pinpointing RNA binding sites and is versatile across various sample types.

Service Contents

Comprehensive RNA-Protein Interaction Mapping Service

Our RNA-Protein Interaction Mapping service delivers precise RNA-protein interaction data across the genome, highlighting both coding and non-coding RNA relationships with proteins. By employing UV crosslinking, we capture and preserve fleeting RNA-protein complexes. These are isolated using our RNA-IP platform, which enriches for target-bound RNA species. Additionally, Seq platform provides in-depth sequencing, pinpointing binding sites down to single nucleotides. This detailed information is vital for understanding post-transcriptional regulation, revealing how interactions affect RNA stability, location, and translation, thus offering insights into cellular functions and disease processes.

Disease-Targeted RNA Analysis Service

Our RNA Analysis service focuses on disease-specific RNA-protein interactions in areas like cancer, neurology, and immunology. It identifies and validates biomarkers and treatment targets, offering critical molecular insights for early-stage research and developing new therapies. Automated library preparation ensures efficient and consistent sample processing, crucial for reliable data. Moreover, we use advanced bioinformatics tools for disease annotation, uncovering disease-related molecular mechanisms. Alongside high-throughput sequencing, our service equips clients with a robust platform for discovering biomarkers and identifying therapeutic targets, giving them an advantage in disease research and therapy development.

Alternative Splicing and Isoform Profiling Service

Our Alternative Splicing and Isoform Profiling service focuses on identifying RNA-binding proteins involved in splicing and comparing transcript variants to get functional insights. It employs high-throughput RT-PCR to find splice sites, advanced bioinformatics tools to make precise calculations, and high-throughput sequencing to cover the transcriptome at scale. This service is necessary for splicing isoform diversity, a fundamental source of knowledge about gene expression regulation, cell fate and tissue-specificity.

Non-Coding RNA and Epitranscriptomics Studies Service

We specialize in examining long non-coding RNAs (lncRNAs), microRNAs (miRNAs), circular RNAs (circRNAs), and RNA modifications such as m6A. We can also undertake to study the interactions with RBPs and gene expression. We detect these RNA modification sites with sophisticated antibodies, ncRNA enrichment kits, mass spectrometry and sequencing equipment. This provides new biomarkers and targets for the actions of these changes in health and disease.

Our Advantages

• Industry-leading CLIP-Seq technology platform : Combines advanced experimental technology with high-precision sequencing platform to ensure data quality and in-depth analysis.
• Comprehensive research support : Offering an all-in-one service from initial experiment planning to final data interpretation.
• Customized solutions : Tailoring the experimental workflow to align with specific project goals and research needs.
• High sensitivity and reliability : Combined with a rigorous quality control process, provide highly reproducible data.

Case Study

Background

This is an investigation using CLIP (crosslinking immunoprecipitation) on interactions between RNA and RBPs. By UV light activating covalent crosslinking between proteins and RNA at their interfaces, small RNA pieces are prepared by UV irradiation and then sonication or nuclease. These clumps break down protein-protein interactions through RNA. The RNA-protein complexes are extracted by immunoprecipitation using specific antibodies and enriched RNA is analyzed with qRT-PCR or RNA-seq.

Methods

The work consists of UV crosslinking, sonication, immunoprecipitation, RNA enrichment and purification, RNA sequencing library preparation and sequencing. Steps: Cell UV crosslinking, sonication, immunoprecipitation, RNA purification, RNA library construction using NEBNext Ultra II RNA Library Prep Kit and high throughput sequencing on an Illumina platform. Analysis: Processing raw data, alignment to genomes/transcriptomes, detecting important binding peak, binding peak structure, functional clustering and motif analysis.

Results

In this project, we performed CLIP-seq experiment for screening interaction rRNA and other RNAs. The bound RNAs were analyzed by Illumina high-throughput sequencing. KEEG biological pathway and GO enrichment analysis of differential peak related genes were conducted to identify the most related genes. Use Igvtools count to compare and analyze the obtained bam file, visualize the bam file, and get the tdf file. Like the CLIP-seq results, the RNA directly bound by the target protein.

Fig. 2. CLIP-seq Result Visualization.

FAQs

• Q: What amount of starting material do you need for a CLIP-Seq experiment?

  A: Usually, 1–10 million cells or 10–50 mg of tissue works. The exact need varies with the target protein and specific experimental details.

• Q: Which sample types are compatible with CLIP-Seq?

  We can work with samples like cultured cells, tissues, and biofluids. Fresh or frozen samples are okay, but keeping RNA intact is key to getting good results.

• Q: Can I use my own antibodies in the immunoprecipitation process?

  Of course. In addition, we can also provide commonly used high-quality antibodies.

• Q: Can CLIP-Seq be customized for specific research needs?

  Absolutely. We provide flexible experimental designs tailored to your target proteins, RNA types, or desired data outputs. Contact us to discuss your specific requirements.

References:

• Hafner M.; et al. CLIP and complementary methods. Nat Rev Methods Primers. 2021;20(1).
• Ule J.; et al. The future of cross-linking and immunoprecipitation (CLIP). Cold Spring Harb Perspect Biol. 2018;10(8):a032243.