PAT-seq - a high-throughput polyadenylation assay for the Illumina platform

Polyadenylation - the cleavage of the nascent mRNA and the addition of a tract consisting of adenine bases in a non-template-dependent manner - is a post-transcriptional modification characteristic of the majority of eukaryotic transcripts. In this study we explore PAT-seq, an Illumina-based poly(A) tail assay, based on the low-throughput G-tailing approach. Briefly, our approach is based on G-tailing poly(A) selected mRNA followed by fragmentation and reverse transcription using a mixture of primers targeting the poly(A)-tail/G-tag junction and the polyadenylation site and the body of the transcripts. The resulting cDNA fragments are sequenced using standard Illumina RNA-seq protocol.

16