Alternation polyadenylation is widespread in eukaryotes, and has demonstrated roles in gene expression regulation. Owing to deep DNA sequencing technologies, global analyses of alternation polyadenylation and their functions have become possible. We present a method to generate poly(A) tags libraries for high-throughput sequencing (PAT-seq). This protocol targets the junction of the 3′-UTR and poly(A) tail of a transcript so it can be positively identified as a poly(A) site. Upon Zinc-mediated limited digestion of total RNA, RNA fragments with poly(A) tail are then isolated and 5′-end repaired. A DNA/RNA hybrid adaptor is ligated to the 5′ end as an anchor. Then the library is generated by reverse transcription with oligo(dT)-adapter followed by PCR amplification. Such a custom poly(A) tags library can be generated from any source poly(A) containing RNA and good for both single- or paired-end sequencing in any Illumina sequencing platforms. This new method has been applied to investigate mRNA polyadenylation in Arabidopsis.
Polyadenylation Illumina sequencing Arabidopsis PAT-seq DNA/RNA hybrid primer
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We thank other lab members for helpful discussion and testing of the protocol. This work was supported by US National Science Foundation (grant nos. IOS–0817829 and IOS-1353354 to QQL), and a grant from Ohio Plant Biotech Consortium.
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