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

• Published in: Methods (San Diego, Calif.) Calif.), 2017-08, Vol.126, p.29-37
• Main Authors: Van Nostrand, Eric L., Shishkin, Alexander A., Pratt, Gabriel A., Nguyen, Thai B., Yeo, Gene W.
• Format: Article
• Language: English
• Subjects: Base Sequence - physiology; Binding Sites - physiology; CLIP-seq; eCLIP-seq; Humans; Reverse Transcription - physiology; RNA binding protein; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Sequence Analysis, RNA - methods
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2017.08.002

•Reverse transcription often terminates or creates deletions at protein-RNA crosslink sites in CLIP-seq.•Altered enzyme and buffer choice can alter crosslink read-through frequency.•Crosslink induced mutation sites (CIMS) are not observed with some reverse transcription conditions. 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 variable rates, indicating that utilization and interpretation of CIMS analysis requires knowledge of the reverse transcriptase enzyme used. Commonly used enzymes, including Superscript III and AffinityScript, show high termination rates in standard magnesium buffer conditions, but show a single base difference in the position of termination for TARDBP motifs. In contrast, manganese-containing buffer promoted read-through at the adduct site. These results validate the use of standard enzymes and also propose alternative enzyme and buffer choices for particularly challenging samples that contain extensive RNA adducts or other modifications that inhibit standard reverse transcription.