PRJNA482397
Monitored eCLIP: high accuracy mapping of RNA-protein interactions
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. However, reverse transcriptases can read through up to one-fourth of the crosslinks leading to false binding site assignments. We developed a “monitored enhanced CLIP” (meCLIP) method to identify read-through cDNAs. A barcoded biotinylated linker is ligated at the 5' end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs that keep the barcode sequence correspond to reverse transcription read-through. Filtering out read-through reads markedly improves the reliability and precision of protein binding sites assignment.