Variation in single-nucleotide sensitivity of eCLIP derived from reverse transcription conditions.

ABSTRACT: Crosslinking and immunoprecipitation (CLIP) followed by high-throughput sequencing identifies the binding sites of RNA binding proteins on RNAs. The covalent RNA-amino acid adducts produced by UV irradiation can cause premature reverse transcription termination and deletions (referred to as crosslink-induced mutation sites (CIMS)), which may decrease overall cDNA yield but are exploited in state-of-the-art CLIP methods to identify these crosslink sites at single-nucleotide resolution. Here, we show the ratio of both crosslinked base deletions and read-through versus termination are highly dependent on the identity of the reverse transcriptase enzyme as well as on buffer conditions used. AffinityScript and TGIRT showed a lack of deletion of the crosslinked base with other enzymes showing va

SUBMITTER: Van Nostrand EL 

PROVIDER: S-EPMC5582984 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature