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Identification of m6A residues at single nucleotide resolution using eCLIP and an accessible custom analysis pipeline

ABSTRACT: Identification of m6A residues at single nucleotide resolution using eCLIP and an accessible custom analysis pipeline

PROVIDER: PRJNA614917 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11403156_1.fastq.gz	Fastqsanger.gz
	SRR11403156_2.fastq.gz	Fastqsanger.gz
	SRR11403158_1.fastq.gz	Fastqsanger.gz
	SRR11403158_2.fastq.gz	Fastqsanger.gz
	SRR11403159_1.fastq.gz	Fastqsanger.gz

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Identification of m6A residues at single nucleotide resolution using eCLIP and an accessible custom analysis pipeline

Project description:We have developed a modified eCLIP-based method (meCLIP) to identify m6A residues at single-nucleotide resolution. By coupling the improvements of eCLIP with an easy-to-use computational pipeline, we have successfully identified over 50,000 unique m6A residues in the two breast cancer cell lines that were analyzed (MCF-7 and MDA-MB-231) and over 8,000 unique residues in HEK-293 cells. We compared these residues to the sites called using the currently most utilized m6A identification method (miCLIP).

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Identification of m<sup>6</sup>A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline.

Project description:Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-m6A antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single-nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.

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Project description:Asterix/Gtsf1 has been previously been shown to be involved in the silencing of retrotransposons in animal germ cells through the piRNA pathway. Using eCLIP and a custom informatic pipeline, we found that Asterix/Gtsf1 binds directly to RNAs and preferentially interacts with tRNAs as a class. Given the role of tRNAs as primers for Long Terminal Repeat (LTR) transposons, this work implicates Asterix/Gtsf1 as exploiting tRNA dependence to identify transposon transcripts and promote piRNA silencing.

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