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High-throughput identification of miRNA targets using CLASH (crosslinking, ligation and sequencing of hybrids)

ABSTRACT: High-throughput identification of miRNA targets using CLASH (crosslinking, ligation and sequencing of hybrids)

PROVIDER: PRJNA217521 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR959751.fastq.gz	Fastqsanger.gz
	SRR959752.fastq.gz	Fastqsanger.gz
	SRR959753.fastq.gz	Fastqsanger.gz
	SRR959754.fastq.gz	Fastqsanger.gz
	SRR959755.fastq.gz	Fastqsanger.gz

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PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) analysis to detect RNA-RNA interaction at poly(A) site region

Project description:In an attempt to discover potential RNA-RNA interactions in mRNA 3’ processing regulation, we developed PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) method, which combines reported CLASH and poly(A) site sequencing protocols. Briefly, HeLa cells were treated with AMT, or without AMT as control, and irradiated at 365 nm for crosslinking. Following nuclear RNA fragmentation and purification, the potential proximate RNAs/PASs were ligated on the oligo d(T)25 beads, hybrids were reverse crosslinked and amplified with the QuantSeq Rev 3' mRNA sequencing library prep kit (Lexogen, Cat. 016.24) and sequenced with Hiseq, producing 150 bp paired end reads.

2019-01-22 | GSE125398 | GEO

High-throughput identification of miRNA targets using CLASH (crosslinking, ligation and sequencing of hybrids)

Project description:Purpose: Identification of miRNA-mRNA interactions in human cells. Method: We used the CLASH technique with PTH-tagged AGO1 protein as a bait. Results: In 6 experiments we identified more than 18,000 miRNA-mRNA interactions in human HEK293 cells, corresponding to targets of 399 miRNAs. Conclusions: The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, non-seed basepairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA basepairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding. 6 samples (E1-E6) were prepared with comparable but not identical protocols, described in more detail in Helwak et al. Cell 2013. Samples E7-E10 were used for the determination of the background of the method resulting from RNA-RNA interactions formed after cell lysis.

2013-08-30 | E-GEOD-50452 | biostudies-arrayexpress

High-throughput identification of miRNA targets using CLASH (crosslinking, ligation and sequencing of hybrids)

2013-08-30 | GSE50452 | GEO

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Project description:We report the identification of potential novel RNA targets for box C/D snoRNAs in human HEK293 cells, using the approaches of UV crosslinking and sequencing of hybrids (CLASH), and formaldehyde crosslinking and sequencing of hybrids (FLASH).

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Modified cross-linking, ligation, and sequencing of hybrids (qCLASH) identifies KSHV microRNA targets in endothelial cells

Project description:KS lesions consist of endothelial cells latently infected with KSHV which express the KSHV miRNAs. Identifying the targets of the KSHV miRNAs will help us understand their role in viral oncogenesis. Cross-Linking and Sequencing of Hybrids (CLASH) is a method for unambiguously identifying miRNA targetomes. We developed a streamlined version of CLASH, called quick CLASH (qCLASH). qCLASH requires a lower initial input of cells than its parent protocol. Additionally, a new fast-growing KSHV-negative endothelial cell line, named TIVE-EX-LTC cells, was established. qCLASH was performed on TIVE-EX-LTC cells latently infected with WT KSHV or a mutant virus lacking miR-K12-11/11*. A number of novel targets of the KSHV miRNAs were identified, including targets of miR-K12-11, the ortholog of cellular oncomiR miR-155. Many of the miRNA targets were involved in processes related to oncogenesis, such as glycolysis, angiogenesis, and cell cycle control.

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Project description:We report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH).

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Project description:Associations between proteins and RNA-RNA duplexes are important in post-transcriptional regulation of gene expression. The CLASH (Cross-linking, Ligation and Sequencing of Hybrids) technique captures RNA-RNA interactions by physically joining two RNA molecules associated with a protein complex into a single chimeric RNA molecule. These events are relatively rare and considerable effort is needed to detect a small number of chimeric sequences amongst millions of non-chimeric cDNA reads resulting from a CLASH experiment. We present the "hyb" bioinformatics pipeline, which we developed to analyse high-throughput cDNA sequencing data from CLASH experiments. Although primarily designed for use with AGO CLASH data, hyb can also be used for the detection and annotation of chimeric reads in other high-throughput sequencing datasets. We examined the sensitivity and specificity of chimera detection in a test dataset using the BLAST, BLAST+, BLAT, pBLAT and Bowtie2 read alignment programs. We obtained the most reliable results in the shortest time using a combination of preprocessing with Flexbar and subsequent read-mapping using Bowtie2. The "hyb" software is distributed under the GNU GPL (General Public License) and can be downloaded from https://github.com/gkudla/hyb.

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The EBV microRNA targetome

Project description:In EBV-associated tumors, such as gastric cancer (GC) and Burkitt’s lymphoma (BL), a high proportion of microRNAs are virally encoded. To explore the targets of both viral and host microRNAs, we performed Crosslinking, Ligation, and Sequencing of Hybrids (CLASH). With this approach, we were able to quantify each Argonaute-bound microRNA-mRNA interaction in a GC (SNU719), and BL (Akata) cell line.

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PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) analysis to detect RNA-RNA interaction at poly(A) site region

Project description:PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) analysis to detect RNA-RNA interaction at poly(A) site region

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