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

ABSTRACT: Ultraviolet crosslinking 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 enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.

SUBMITTER: Lorenz DA

PROVIDER: S-EPMC9834051 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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

Lorenz Daniel A DA Her Hsuan-Lin HL Shen Kylie A KA Rothamel Katie K Hutt Kasey R KR Nojadera Allan C AC Bruns Stephanie C SC Manakov Sergei A SA Yee Brian A BA Chapman Karen B KB Yeo Gene W GW

Nature methods 20221222 1

Ultraviolet crosslinking 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 enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protecte ...[more]

PMID: 36550273

Similar Datasets

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

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

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

| PRJNA846294 | ENA

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.

| S-EPMC4887338 | biostudies-literature

Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq.

Project description:Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false positive rates. Here, we present the R/Bioconductor package DEWSeq that makes use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing. The improvement not only relates to the number of binding sites (3.1-fold with known motifs for RBFOX2), but also their subcellular localization (1.9-fold of mitochondrial genes for FASTKD2) and structural targets (2.2-fold increase of stem-loop regions for SLBP. On several orthogonal CLIP-seq datasets, DEWSeq recovers a larger number of motif-containing binding sites (3.3-fold). DEWSeq is a well-documented R/Bioconductor package, scalable to adequate numbers of replicates, and tends to substantially increase the proportion and total number of RBP binding sites containing biologically relevant features.

| S-EPMC10783507 | biostudies-literature

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

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

Project description:This SuperSeries is composed of the SubSeries listed below.

2016-03-28 | GSE77634 | GEO

Discovery of DAP3-binding RNA sites by eCLIP

Project description:eCLIP paired-end sequencing of DAP3 in EC109 cells with two biological replicates. This analysis revealed the genome-wide RNA binding landscape of DAP3, as the supporting data of the main publication.

2020-06-23 | GSE144318 | GEO

Discovery of DAP3-binding RNA sites by eCLIP

Project description:Discovery of DAP3-binding RNA sites by eCLIP

| PRJNA603360 | ENA

Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces.

Project description:Discovering the interaction mechanism and location of RNA-binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts compared to protein-absent transcripts in four human cell lines to identify transcriptome-wide footprints (fSHAPE) on RNA. Structural analyses indicate that fSHAPE precisely detects nucleobases that hydrogen bond with protein. We demonstrate that fSHAPE patterns predict binding sites of known RBPs, such as iron response elements in both known loci and previously unknown loci in CDC34, SLC2A4RG, COASY, and H19. Furthermore, by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs, we interrogate specific RNA-protein complexes, such as histone stem-loop elements and their nucleotides that hydrogen bond with stem-loop-binding proteins. Together, these technologies greatly expand our ability to study and understand specific cellular RNA interactions in RNA-protein complexes.

| S-EPMC8074864 | biostudies-literature

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