3' End Sequencing Library Preparation with A-seq2

Martin, Georges and Schmidt, Ralf and Gruber, Andreas J. and Ghosh, Souvik and Keller, Walter and Zavolan, Mihaela. (2017) 3' End Sequencing Library Preparation with A-seq2. Journal of Visualized Experiments (128). e56129.

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Official URL: http://edoc.unibas.ch/56968/

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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 10(6) or fewer cells.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Computational & Systems Biology > Bioinformatics (Zavolan)
UniBasel Contributors:Zavolan, Mihaela and Martin, Georges and Gruber, Andreas J. and Schmidt, Ralf and Ghosh, Souvik and Keller, Walter
Item Type:Article, refereed
Article Subtype:Research Article
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:16 May 2018 06:48
Deposited On:16 May 2018 06:48

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