Chapter title |
Polyadenylation Site-Based Analysis of Transcript Expression by 3′READS+
|
---|---|
Chapter number | 6 |
Book title |
mRNA Processing
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7204-3_6 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7203-6, 978-1-4939-7204-3
|
Authors |
Dinghai Zheng, Bin Tian |
Abstract |
Deep sequencing of the 3' end region of poly(A)+ RNA identifies the cleavage and polyadenylation site (PAS) and measures transcript abundance. However, mispriming at internal A-rich regions by the oligo-dT oligo in reverse transcription can lead to falsely identified PASs. This problem can be resolved by direct ligation of an adapter to the 3' end of RNA. However, ligation-based methods are often inefficient. Here, we describe 3'READS+, an accurate and sensitive method for deep sequencing of the 3' end of poly(A)+ RNA. Through partial digestion by RNase H of the poly(A) tail bound to a locked nucleic acid (LNA)/DNA hybrid oligo, this method sequences an optimal number of terminal A's, which balances sequencing quality and accurate identification of PAS in A-rich regions. With efficient ligation steps, 3'READS+ is amenable to small amounts of input RNA. 3'READS+ can also be readily used as a cost-effective method for gene expression analysis. |
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