title: Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq.
abstract: Next-generation sequencing has great potential for application in bacterial transcriptomics. However, unlike eukaryotes, bacteria have no clear mechanism to select mRNAs over rRNAs; therefore, rRNA removal is a critical step in sequencing-based transcriptomics. Duplex-specific nuclease (DSN) is an enzyme that, at high temperatures, degrades duplex DNA in preference to single-stranded DNA. DSN treatment has been successfully used to normalize the relative transcript abundance in mRNA-enriched cDNA libraries from eukaryotic organisms. In this study, we demonstrate the utility of this method to remove rRNA from prokaryotic total RNA. We evaluated the efficacy of DSN to remove rRNA by comparing it with the conventional subtractive hybridization (Hyb) method. Illumina deep sequencing was performed to obtain transcriptomes from Escherichia coli grown under four growth conditions. The results clearly showed that our DSN treatment was more efficient at removing rRNA than the Hyb method was, while preserving the original relative abundance of mRNA species in bacterial cells. Therefore, we propose that, for bacterial mRNA-seq experiments, DSN treatment should be preferred to Hyb-based methods.
authors: Institute of Molecular Biology and Genetics, School of Biological Sciences & Institute of Bioinformatics (BIOMAX), Seoul National University, Seoul, Republic of Korea.
authors: Hana
authors: H
authors: Yi
authors: Yong-Joon
authors: YJ
authors: Cho
authors: Sungho
authors: S
authors: Won
authors: Jong-Eun
authors: JE
authors: Lee
authors: Hyung
authors: H
authors: Jin Yu
authors: Sujin
authors: S
authors: Kim
authors: Gary P
authors: GP
authors: Schroth
authors: Shujun
authors: S
authors: Luo
authors: Jongsik
authors: J
authors: Chun
mesh_terms: D003851:Deoxyribonucleases; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D003851:Deoxyribonucleases