Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins

ABSTRACT: Identification of in vivo direct RNA targets for RNA binding proteins (RBP) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus. Further, we show that TAG-eCLIP performed using tag-specific antibodies can yield the same robust binding profiles after proper control normalization as eCLIP with antibodies against endogenous proteins. Finally, we note that antibodies against commonly used tags can immunoprecipitate significant amounts of antibody-specific RNA, emphasizing the need for paired controls alongside each experiment for normalization. TAG-eCLIP enables eCLIP profiling of new native proteins where no suitable antibody exists, expanding the RBP-RNA landscape.

ORGANISM(S): Homo sapiens

PROVIDER: GSE88722 | GEO | 2017/08/22

SECONDARY ACCESSION(S): PRJNA348371

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Deciphering RBP - alternative splicing networks in ALS iPSC-MN: TDP-43, NOVA1, NOVA2 and RBFOX2 eCLIP-seq

Project description:We produced enhanced cross-linking immunoprecipitation (eCLIP) sequencing data of the RNA binding proteins (RBP) TDP-43, NOVA1, NOVA2 and RBFOX2 in iPSC motor neurons of two healthy control individuals, respectively.

| EGAS00001005880 | EGA

Homo sapiens

Project description:CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins

| PRJNA348371 | ENA

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

Best practices for eCLIP experiments and analysis [poor quality]

Project description:Enhanced cross-linking immunoprecipitation (eCLIP) featuring a size-matched input control has been recently applied to profile the binding sites of more than one hundred RNA binding proteins (RBPs). However computational pipelines and quality control metrics needed to process CLIP data at scale have yet to be well defined. Here, we describe our ENCODE eCLIP processing pipeline (https://github.com/YeoLab/eclip), enabling users to go from raw reads to processed peaks that are enriched above paired input, reproducible across biological replicates, and can be directly compared against the public ENCODE eCLIP resource. In particular, we discuss processing steps designed to address common artifacts, including properly quantifying unique RNA fragments bound by both unique genomic- and repetitive element-mapped reads. Using manual quality annotation of 350 ENCODE eCLIP experiments, we develop metrics for quality assessment of eCLIP experiments prior to and after sequencing, including library yield, number of unique fragments in the library, total binding relative information, and biological reproducibility. In particular, we quantify the commonly believed linkage between depth of sequencing and peak discovery, and derive methods for estimating required sequencing depth based on pre-sequencing metrics. Finally we provide recommendations for the common question of integrating RBP binding information with RNA-seq to generate splicing maps representing the positional effect of binding on alternative splicing. These pipelines and QC metrics enable large-scale processing and analysis of eCLIP data, and will help to standardize rigorous analysis of RBP binding data.

2018-06-08 | GSE107767 | GEO

Best practices for eCLIP experiments and analysis [uncertain quality]

2018-06-08 | GSE107766 | GEO

Profiling of RNA-binding protein binding sites by in-situ reverse transcription-based sequencing

Project description:RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA substrates are still lacking, especially when the interaction is dynamic or materials are limited. Here we present an assay of reverse transcription-based RBP binding sites sequencing (ARTR-seq), which relies on in-situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet cross-linking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic binding of RBPs over a short period of time, as demonstrated by the discovery of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 min.

2023-11-05 | GSE226161 | GEO

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

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 | GSE77633 | GEO

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

2016-03-28 | GSE77629 | GEO

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

2016-03-28 | GSE77339 | GEO

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

2016-04-07 | GSE80039 | GEO