Detection of Reverse Transcriptase Termination Sites Using cDNA Ligation and Massive Parallel Sequencing

  • Lukasz J. Kielpinski
  • Mette Boyd
  • Albin Sandelin
  • Jeppe Vinther
Part of the Methods in Molecular Biology book series (MIMB, volume 1038)


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 words

Reverse 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.


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Lukasz J. Kielpinski
    • 1
  • Mette Boyd
    • 1
    • 2
  • Albin Sandelin
    • 1
    • 2
  • Jeppe Vinther
    • 1
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagenDenmark
  2. 2.Biotech Research and Innovation CentreUniversity of CopenhagenCopenhagenDenmark

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