Single-molecule polyadenylated tail sequencing (SM-PAT-Seq) to measure polyA tail lengths transcriptome-wide

Polyadenylation of the 3′ end of mRNAs is an important mechanism for regulating their stability and translation. We developed a nucleotide-resolution, transcriptome-wide, single-molecule SM-PAT-Seq method to accurately measure the polyA tail lengths of individual transcripts using long-read sequencing. The method generates cDNA using a double stranded splint adaptor targeting the far 3′ end of the polyA tail for first strand synthesis along with random hexamers for second strand synthesis. This straight-forward method yields accurate polyA tail sequence lengths, can identify non-A residues in those tails, and quantitate transcript abundance.