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Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP [dataset 2]

ABSTRACT: RNA-binding proteins are instrumental for post-transcriptional gene regulation, yet transcriptome-wide methods to profile RNA-protein interactions remain technically challenging. We present an improved library preparation strategy for cross-linking and immunoprecipitation (CLIP) that involves tailing and ligation of cDNA molecules (TLC) for increased sensitivity and efficiency. TLC-CLIP eliminates time-consuming purifications, reduces sample loss, and minimises experimental steps, allowing precise profiling of RNA-protein interactions from limited starting material at nucleotide resolution.

ORGANISM(S): Homo sapiens

PROVIDER: GSE225357 | GEO | 2023/05/10

REPOSITORIES: GEO

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Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP [dataset 1]

Project description:RNA-binding proteins are instrumental for post-transcriptional gene regulation, yet transcriptome-wide methods to profile RNA-protein interactions remain technically challenging. We present an improved library preparation strategy for cross-linking and immunoprecipitation (CLIP) that involves tailing and ligation of cDNA molecules (TLC) for increased sensitivity and efficiency. TLC-CLIP eliminates time-consuming purifications, reduces sample loss, and minimises experimental steps, allowing precise profiling of RNA-protein interactions from limited starting material at nucleotide resolution.

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Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP

Project description:This SuperSeries is composed of the SubSeries listed below. GSE200432 - TLC-CLIP data GSE225357 - RNA-Seq data

2023-05-10 | GSE225358 | GEO

Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP

Project description:Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP

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GoldCLIP: Gel-omitted Ligation-dependent CLIP

Project description:Protein-RNA interaction networks are essential to understand gene regulation control. Identifying the binding sites of RNA-binding proteins (RBPs) by CLIP (UV-crosslinking and immunoprecipitation) represents one of the most powerful methods to map in vivo protein-RNA interactions. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allowed us to perform highly reproducible CLIP experiments with classical RBP such as PTB in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse factors to uncover their endogenous targets.

2018-05-20 | GSE111406 | GEO

Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts.

Project description:High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. Up to 45% of peaks in publicly available HITS-CLIP libraries are attributable to this artifact, and the majority of libraries have detectable levels of mispriming. We also found that standard techniques for validating miRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks. Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact. Argonaute HITS-CLIP on the MCF-7 breast cancer cell line treated with 17Î²-estradiol for 0, 6 or 24 hours using a nested reverse transcriptions pimer and protected or unprotected reverse PCR primers for library amplification.

2016-02-19 | E-GEOD-78059 | biostudies-arrayexpress

Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts.

2016-02-19 | GSE78059 | GEO

Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP [dataset 2]

Project description:Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP [dataset 2]

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Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP [dataset 1]

Project description:Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP [dataset 1]

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Implementation of PAR-CLIP to characterize RNA-protein interactions in prokaryotes at nucleotide resolution

Project description:The identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as Crosslinking and Immunoprecipitation (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP) in vivo. Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA and for the isolation of RNA crosslinked to RNA-BPs of interest. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes.

2022-10-18 | MSV000090547 | MassIVE

A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol

Project description:Streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. Protocol is based on direct ligation of a fluorescently labeled adapter to the 3’end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation time from 4-5 to 2 days and increases sensitivity by 10-100-fold.

2020-12-29 | GSE163925 | GEO

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