1 Computational and RNA Biology, Department of Biology, Faculty of Science, Københavns Universitet2 BRIC distribution, BRIC, Københavns Universitet3 Department of Biology, Faculty of Science, Københavns Universitet4 Department of Biology, Faculty of Science, Københavns Universitet5 Computational and RNA Biology, Department of Biology, Faculty of Science, Københavns Universitet
Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR amplification, Illumina adapters and index sequences are introduced, thereby allowing amplicons to be pooled and sequenced on the standard Illumina platform for genomic DNA sequencing. Moreover, we demonstrate how to map sequencing reads and perform analysis of the sequencing data with freely available tools that do not require formal bioinformatics training. As an example, we apply the method to detection of transcription start sites in mouse liver cells.
Methods in Molecular Biology (clifton, N.j.), 2013, Vol 1038, p. 213-31