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3′ End Sequencing Library Preparation with A-seq2

JoVE Journal
Biology

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JoVE Journal Biology

3′ End Sequencing Library Preparation with A-seq2

3′ End Sequencing Library Preparation with A-seq2

DOI:

10.3791/56129-v

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12:01 min

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October 10, 2017

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Georges Martin, Ralf Schmidt, Andreas J. Gruber, Souvik Ghosh, Walter Keller, Mihaela Zavolan²

¹Computational and Systems Biology, Biozentrum,University of Basel, ²Swiss Institute of Bioinformatics, Biozentrum,University of Basel

Chapters

00:05Title
00:46Isolation of mRNA from Cells
03:245′ End Phosphorylation, DNase Treatment, and 3′ End Blocking
04:30Ligation of Reverse 3′ Adapters to 5′ End of RNA Fragments and Reverse Transcription
05:59Digestion with Uracil DNA Glycosylase Enzyme Mix and Ligation of 5′ Adapters to 5′ Ends of cDNA
07:44Pilot PCR, Amplification of Libraries, and Size Selection
09:21Computational Analysis of DNA Sequence Data
10:03Results: The A-seq2 Method Maps pre-mRNA 3′ End Processing Sites
10:52Conclusion

Summary

Automatic Translation

This protocol describes a method for mapping pre-mRNA 3' end processing sites.

Tags

Keywords: 3′ End Sequencing A-seq2 MRNA Processing Polyadenylation Transcript Isoforms Cell Types Poly(A) Stretches Adapter Dimers Cell Lysis Oligo D(T)25 Magnetic Beads Buffer A Buffer B Alkaline Hydrolysis RNA Isolation Polynucleotide Kinase

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