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.
Alternative cleavage and polyadenylation Deep sequencing RNA-seq 3′ end sequencing 3′READS+
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
Fu Y et al (2011) Differential genome-wide profiling of tandem 3′ UTRs among human breast cancer and normal cells by high-throughput sequencing. Genome Res 21(5):741–747CrossRefPubMedPubMedCentralGoogle Scholar
Jan CH et al (2011) Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs. Nature 469(7328):97–101CrossRefPubMedGoogle Scholar
Hoque M et al (2013) Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing. Nat Methods 10(2):133–139CrossRefPubMedGoogle Scholar
Nam DK et al (2002) Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription. Proc Natl Acad Sci U S A 99(9):6152–6156CrossRefPubMedPubMedCentralGoogle Scholar
Lee JY, Park JY, Tian B (2008) Identification of mRNA polyadenylation sites in genomes using cDNA sequences, expressed sequence tags, and trace. Methods Mol Biol 419:23–37CrossRefPubMedGoogle Scholar
Li W et al (2015) Systematic profiling of poly(A)+ transcripts modulated by core 3′ end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation. PLoS Genet 11(4):e1005166CrossRefPubMedPubMedCentralGoogle Scholar