A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq

RNA-sequencing is a powerful and increasingly prevalent method to answer biological questions. Depletion of ribosomal RNA (rRNA), which accounts for 80% of total RNA, is an extremely important step to increase the power of RNA-seq. Selection for polyadenylated RNA is a commonly used approach that excludes rRNA, as well as, important non-polyadenylated RNAs, such as histones, circular RNAs, and many long noncoding RNAs. Commercial methods to deplete rRNA are cost-prohibitive and the gold standard method is no longer available as a standalone kit. Alternative non-commercial methods suffer from inconsistent depletion. Through careful characterization of all reaction parameters, we developed an optimized RNaseH-based depletion of human rRNA. Our method exhibited comparable or better rRNA depletion compared to commercial kits at a fraction of the cost and across a wide-range of input RNA amounts.