The RNA Interactome Toolkit
The RNA core allows for the investigation of multiple aspects of RNA interactomics. Through cutting-edge technology and streamlined sets of protocols/workflows, our team can usher in the next frontier of RNA biology- from multiplexed binding profiles from human tissues to high-throughput functional RNA drug screens.
Enhanced CLIP-sequencing (eCLIP-seq)
eCLIP: Mapping RNA-protein interactions transcriptome-wide
eCLIP
We developed enhanced crosslinking and immunoprecipitation (eCLIP)1,2 to map the transcriptome-wide binding sites of RNA-binding proteins (RBPs). RBP-RNA maps detail the occupancy of proteins on RNA at nucleotide-level resolution, providing incredibly powerful insights into the regulation of RNA processing.
Ribo-eCLIP
Applied to ribosomal proteins, eCLIP measures ribosome occupancy (ribo-eCLIP) and -- unlike ribosome profiling (ribo-seq) -- can distinguish mRNA targets of ribosomes with different protein composition. Ribo-eCLIP unveils diverse mediators and perturbations of translation, giving mechanistic insight into mRNA translation efficiency and global translation landscape.
OligoCLIP
Our OligoCLIP3 method simultaneously captures up to 10 RBPs from a single sample, which allows profiling multiple RBPs even when sample amounts are limiting. We continue to develop highly parallelized workflows for the rapid and systematic mapping of RBP-RNA protein networks in disease-relevant systems.
- Van Nostrand et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nature Methods (2016).
- Van Nostrand et al. Robust, cost-effective profiling of RNA binding protein targets with single-end enhanced crosslinking and immunoprecipitation (seCLIP). Methods in Molecular Biology (2017).
- Lorenz et al. Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP. Nature Methods (2023).
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Circular RNA sequencing (circRNA-seq)
circRNA: studying the multifaceted role of circular RNA
Circular RNAs (circRNAs) are generated when pre-mRNAs are spliced in reversed order ('backspliced'). They play multifaceted functions as microRNA sponges, protein decoys, and protein scaffolds. They also recruit translation machinery and act as templates for translation. CircRNAs play critical roles in many biological contexts including cancer, immunology, neuroscience, and development. Due to their relatively higher stability compared to linear RNA molecules, circRNAs are becoming increasingly recognized as potential biomarkers in disease. The RNA Center offers cutting-edge circRNA sequencing technologies to empower novel biomarker discovery and circRNA biology research.
Small-scale circRNA sequencing
We have developed a novel circRNA sequencing library preparation protocol (manuscript in preparation) that is orders of magnitude more efficient that previous methods, allowing for highly sensitive detection of circRNAs from as little as 50 ng of total RNA, with comparable performance as published methods that typically require >1µg RNA. This makes it possible to study precious/limiting samples such as clinical specimens and cells isolated by FACS.
Long read circRNA sequencing
Bioinformatic identification of circRNAs is heavily based on detection of the backsplicing junctions that are generated during biogenesis. As a result, alternative splicing information of the circRNAs is lost in short-read sequencing. circRNA sequencing using Nanopore long-read instrumentation allows for sequencing of the entire RNA circle, thus sequenced. This complete sequence information allows for splicing regulatory mechanisms to be discovered and provides much deeper insight into the potential function of each circRNA species.
CircRNA interactome assays
We are developing novel tools to study RBP-circRNA interactions and circRNA translation. Contact us for more information.
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Protein production
Custom Protein Expression and Purification