TRENDseq-A highly multiplexed high throughput RNA 3' end sequencing for mapping alternative polyadenylation

Alternative polyadenylation (APA) is a widespread and highly dynamic mechanism of gene regulation. It affects more than 70% of all genes, resulting in transcript isoforms with distinct 3' end termini. APA thereby considerably expands the diversity of the transcriptome 3' end (TREND). This leads to mRNA isoforms with profoundly different physiological effects, by affecting protein output, production of distinct protein isoforms, or modulating protein localization. APA is globally regulated in various conditions, including developmental and adaptive programs. Since perturbations of APA can disrupt biological processes, ultimately resulting in most devastating disorders, querying the APA landscape is crucial to decipher underlying mechanisms, resulting consequences and potential diagnostic and therapeutic implications. Here we provide a detailed step-by-step protocol for TRENDseq, a method for transcriptome-wide high-throughput sequencing of polyadenylated RNA 3' ends in a highly multiplexed fashion. TRENDseq exploits linear amplification of the starting material to improve sensitivity while significantly reducing the amount of input material. It thereby represents a powerful tool to study APA in numerous experimental set-ups and/or limited human samples in a highly multiplexed and reproducible manner.