Polyadenylation site-based analysis of transcript expression by 3′READS+

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.