RPAD (RNase R treatment, polyadenylation, and poly(A)+ RNA depletion) method to isolate highly pure circular RNA

Abstract Recent developments in high-throughput RNAsequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ) RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAswith potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins(RBPs). The biochemical enrichment of circRNAs by exoribonucleasetreatment or by depletion of polyadenylated RNAscoupled with deep-sequencingis widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently non- polyadenylatedand highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial R Nase R treatment followed by P olyadenylation and poly( A ) + RNAD epletion. These joint interventions drastically depleted linear RNAsleading to isolation of highly pure circRNAs from total RNApools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.