Poly(A)-ClickSeq: Poly(A)-Primed Protocol with Single Indexing using Poly(A)-ClickSeq Kit v1

Abstract

Poly(A)-ClickSeq is a library preparation method used to target the 3’ ends of polyadenylated RNA, such as eukaryotic mRNAs. This technique offers an alternative to conventional RNA-seq methods that provide the user with sequencing reads that cover entire transcripts. Instead, the 3’ end targeting protocol of Poly(A)-ClickSeq enables a more cost efficient and straightforward method for measuring differential gene expression and simultaneously the mapping of poly(A) sites which can be used for alternative polyadenylation studies. The process takes advantage of the chain-terminating properties of 3′-azido-nucleotides, which are included the initial in vitro reverse-transcription reactions uniformly required for RNAseq. In Poly(A)-ClickSeq (PAC-Seq), priming occurs from poly(A)-tails using an unanchored oligo-dT primer and only AzATP, AzGTP and AzCTP (collectively known as AzVTPs) are supplemented in the RT reaction. As a result, cDNA synthesis does not terminate in the poly(A)-tail, but rather continues until the 3’UTR is reached. Thereafter, the modified nucleotides (AzVTPs) are stochastically incorporated into the nascent cDNA at a programmable distance upstream of the 3’UTR/Poly(A)-tail junction, yielding cDNA fragments blocked at their 3′ends with azido groups. The 3′-azido-blocked cDNA fragments are ‘click-ligated’ onto alkyne-functionalized sequencing adaptors, which can subsequently be PCR-amplified to yield a sequencing-ready NGS library. PAC-Seq offers unique advantages over common RNA sequencing and 3’end mapping protocols in that it does not require the purification, selection, or fragmentation steps typically required in RNA-seq approaches. Sample preparation is started directly from crude total cellular RNA. Furthermore, click-chemistry is utilized to attach the required sequencing adapter, rather than commonly-used enzymatic reactions. Overall, this results in increased efficiency of the protocol, fewer processing steps, and reduced time from RNA to sequencing-ready libraries.