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

PLoS Genet. 2021 Sep 27;17(9):e1009821. doi: 10.1371/journal.pgen.1009821. eCollection 2021 Sep.


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.

Grant support

This study is supported by the Korea Bio Grand Challenge (2018M3A9H3024759) to BKC, C1 Gas Refinery Program (2018M3D3A1A01055733) to BKC, and the National Research Foundation of Korea grant (2021R1A2C1012589) to SC through National Research Foundation of Korea (https://www.nrf.re.kr/eng/index) funded by the Ministry of Science and ICT, Republic of Korea. This work was also supported by a grant from the Novo Nordisk Fonden (NNF10CC1016517) (https://novonordiskfonden.dk/en/) to BP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.