Differential regulation of alternatively polyadenylated isoforms

Alternative Cleavage and Polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we employed a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This comparison allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape the APA profiles in the subcellular locations. Subsequently, we depleted these cells of Dicer to compromise the miRNA biogenesis pathway to isolate the differentially regulated APA events that were regulated by miRNA. Overall design: Two biological replicates of Dicer-depleted and non-depleted HEK293 subcellular fractions were analysed using 3''region extraction and deep sequencing to quantitatively measure the usage of each cleavage and polyadenylation site transcriptome-wide. In addition, 3''READS was performed on the subcellular RNA fractions of HBL melanocyte cells to see if the pattern of differential APA regulation holds between cell lines. Furthermore, standard RNA-seq was performed using the Ion Proton system on HEK293 subcellular RNA fractions. Please note that the raw data from two replicates of Dicer-depleted and non-depleted HEK293 subcellular fractions samples were merged to generate processed data, which is linked to the first replicate sample record (i.e. *rep1.1). For example, the processed data files, C0d.p.bw and C0d.m.bw, were generated from both 3''READs cyto_rep1.1 and 3''READs cyto_rep1.2 sample data and linked to the ''3''READs cyto_rep1.1'' sample records as supplementary data file.