RiboRid: A low cost, advanced, and ultra-efficient method to remove ribosomal RNA for bacterial transcriptomics.

RNA sequencing techniques have enabled the systematic elucidation of gene expression (RNA-Seq), transcription start sites (differential RNA-Seq), transcript 3′ ends (Term-Seq), and post-transcriptional processes (ribosome profiling). The main challenge of transcriptomic studies is to remove ribosomal RNAs (rRNAs), which comprise more than 90% of the total RNA in a cell. Here, we report a low-cost and robust bacterial rRNA depletion method, RiboRid, based on the enzymatic degradation of rRNA by thermostable RNase H. This method implemented experimental considerations to minimize nonspecific degradation of mRNA and is capable of depleting pre-rRNAs that often comprise a large portion of RNA, even after rRNA depletion. We demonstrated the highly efficient removal of rRNA up to a removal efficiency of 99.99% for various transcriptome studies, including RNA-Seq, Term-Seq, and ribosome profiling, with a cost of approximately $10 per sample. This method is expected to be a robust method for large-scale high-throughput bacterial transcriptomic studies. Author summary: Removal of ribosomal RNAs, a major constituent (over 90%) of cellular RNA is a critical experimental step for transcriptomic studies that deal with messenger RNAs. In this manuscript, we describe a robust method to subtract ribosomal RNA from various RNA samples. The method is based on the enzymatic degradation of target RNA by short complementary DNA and RNA:DNA duplex specific nuclease. The method comprises carefully designed experimental procedures to minimize experimental bias and unwanted removal of messenger RNAs. We validate the method on various types of transcriptomic studies for seven diverse bacterial species. This method successfully removed ribosomal RNA with over 99% of efficiency and it was comparable to commercial systems even for degraded RNA samples at a fraction of a cost. [ABSTRACT FROM AUTHOR]