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Monitored eCLIP: high accuracy mapping of RNA-protein interactions.

ABSTRACT: CLIP-seq methods provide transcriptome-wide snapshots of RNA-protein interactions in live cells. Reverse transcriptases stopping at cross-linked nucleotides sign for RNA-protein binding sites. Reading through cross-linked positions results in false binding site assignments. In the 'monitored enhanced CLIP' (meCLIP) method, a barcoded biotinylated linker is ligated at the 5' end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs keeping the barcode sequence correspond to reverse transcription read-throughs. Read through occurs in unpredictable proportions, representing up to one fourth of total reads. Filtering out those reads strongly improves reliability and precision in protein binding site assignment.

SUBMITTER: Hocq R

PROVIDER: S-EPMC6265473 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Monitored eCLIP: high accuracy mapping of RNA-protein interactions.

Hocq Rémi R Paternina Janio J Alasseur Quentin Q Genovesio Auguste A Le Hir Hervé H

Nucleic acids research 20181101 21

CLIP-seq methods provide transcriptome-wide snapshots of RNA-protein interactions in live cells. Reverse transcriptases stopping at cross-linked nucleotides sign for RNA-protein binding sites. Reading through cross-linked positions results in false binding site assignments. In the 'monitored enhanced CLIP' (meCLIP) method, a barcoded biotinylated linker is ligated at the 5' end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs keeping the barcode sequence corr ...[more]

PMID: 30252095

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