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3' End Sequencing Library Preparation with A-seq2

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2017

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Martin, Georges
Schmidt, Ralf
Ghosh, Souvik
Keller, Walter
Zavolan, Mihaela

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https://doi.org/10.3791/56129
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Journal of Visualized Experiments. JoVE. 2017(128), e56129. eISSN 1940-087X. Available under: doi: 10.3791/56129

Zusammenfassung

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3' untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3'UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3' end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3' end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3' ends in a wide range of conditions, from 106 or fewer cells.

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570 Biowissenschaften, Biologie

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Molecular Biology, Issue 128, Polyadenylation, poly(A) site, pre-mRNA 3' end sequencing, A-seq2, data analysis software, deep sequencing

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ISO 690MARTIN, Georges, Ralf SCHMIDT, Andreas J. GRUBER, Souvik GHOSH, Walter KELLER, Mihaela ZAVOLAN, 2017. 3' End Sequencing Library Preparation with A-seq2. In: Journal of Visualized Experiments. JoVE. 2017(128), e56129. eISSN 1940-087X. Available under: doi: 10.3791/56129
BibTex
@article{Martin2017-10-10Seque-50941,
  year={2017},
  doi={10.3791/56129},
  title={3' End Sequencing Library Preparation with A-seq2},
  number={128},
  journal={Journal of Visualized Experiments},
  author={Martin, Georges and Schmidt, Ralf and Gruber, Andreas J. and Ghosh, Souvik and Keller, Walter and Zavolan, Mihaela},
  note={Article Number: e56129}
}
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