Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

TY - JOUR

T1 - Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

AU - Kielpinski, Lukasz J

AU - Boyd, Mette

AU - Sandelin, Albin

AU - Vinther, Jeppe

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - Animals

KW - Bacteriophages

KW - DNA, Complementary

KW - High-Throughput Nucleotide Sequencing

KW - Mice

KW - Polymerase Chain Reaction

KW - RNA Ligase (ATP)

KW - RNA-Directed DNA Polymerase

KW - Reverse Transcription

KW - Sequence Analysis, DNA

U2 - 10.1007/978-1-62703-514-9_13

DO - 10.1007/978-1-62703-514-9_13

M3 - Journal article

C2 - 23872978

SN - 1064-3745

VL - 1038

SP - 213

EP - 231

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

ER -