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Rivista
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Biologia
/
3′ End Sequencing Library Preparation with A-seq2
JoVE Journal
Biologia
This content is Free Access.
JoVE Journal
Biologia
3′ End Sequencing Library Preparation with A-seq2
3′ End Sequencing Library Preparation with A-seq2
DOI:
10.3791/56129-v
•
12:01 min
•
October 10, 2017
•
Georges Martin
,
Ralf Schmidt
,
Andreas J. Gruber
,
Souvik Ghosh
,
Walter Keller
,
Mihaela Zavolan
2
1
Computational and Systems Biology, Biozentrum
,
University of Basel
,
2
Swiss Institute of Bioinformatics, Biozentrum
,
University of Basel
Capitoli
00:05
Titolo
00:46
Isolation of mRNA from Cells
03:24
5′ End Phosphorylation, DNase Treatment, and 3′ End Blocking
04:30
Ligation of Reverse 3′ Adapters to 5′ End of RNA Fragments and Reverse Transcription
05:59
Digestion with Uracil DNA Glycosylase Enzyme Mix and Ligation of 5′ Adapters to 5′ Ends of cDNA
07:44
Pilot PCR, Amplification of Libraries, and Size Selection
09:21
Computational Analysis of DNA Sequence Data
10:03
Results: The A-seq2 Method Maps pre-mRNA 3′ End Processing Sites
10:52
Conclusion
Summary
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This protocol describes a method for mapping pre-mRNA 3' end processing sites.
Tags
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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