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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Binding Sites
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Cross-Linking Reagents / chemistry*
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DEAD-box RNA Helicases / genetics
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DEAD-box RNA Helicases / metabolism
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DNA, Complementary
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Eukaryotic Initiation Factor-4A / genetics
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Eukaryotic Initiation Factor-4A / metabolism
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Humans
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Immunoprecipitation / methods*
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Oligonucleotides / chemistry
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Oligonucleotides / genetics
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Proteins / chemistry
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Proteins / genetics
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Proteins / metabolism*
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RNA / metabolism*
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RNA Helicases / genetics
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RNA Helicases / metabolism
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Reverse Transcription
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Trans-Activators / genetics
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Trans-Activators / metabolism
Substances
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Cross-Linking Reagents
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DNA, Complementary
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Oligonucleotides
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Proteins
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Trans-Activators
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RNA
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Eukaryotic Initiation Factor-4A
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EIF4A3 protein, human
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DEAD-box RNA Helicases
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RNA Helicases
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UPF1 protein, human