Detection of Reverse Transcriptase Termination Sites Using cDNA Ligation and Massive Parallel Sequencing
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.
Key wordsReverse transcription Termination Sequencing TS2l26 RNA ligase CAGE Galaxy
The research was funded by the Danish Council for Strategic Research, the Lundbeck Foundation and the Novo Nordisk Foundation. Morten Lindow and Susanna Obad, Santaris Pharma, provided mouse liver samples and RIKEN/Piero Carninci provided the updated CAGE protocol as well as advice ahead of publication.
- 16.Blankenberg D, Von Kuster G, Coraor N et al. (2010) Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol Chapter 19:Unit 19.10.11–21Google Scholar
- 18.Hannon-Lab, Gordon A (2010) FASTX-toolkit: FASTQ/A short-reads pre-processing tools. http://hannonlab.cshl.edu/fastx_toolkit/
- 19.R Foundation for Statistical Computing (2012) R: A language and environment for statistical computing, 2151st edn. R Foundation for Statistical Computing, Vienna, AustriaGoogle Scholar