Construction of prokaryotic strand-specific primary-transcripts saturated RNASeq library by controlled heat magnesium-dependent mRNA degradation Kirill S. Mironov a, * , Maria Shumskaya b , Dmitry A. Los a a Department of Molecular Biosystems, K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, 127276, Moscow, Russia b Department of Biology, School of Natural Sciences, Kean University,1000 Morris Ave, Union, NJ, 07083, USA article info Article history: Received 5 June 2020 Received in revised form 26 July 2020 Accepted 2 August 2020 Available online 14 August 2020 Keywords: RNASeq cDNA library preparation mRNA Primary transcripts XRN-1 rRNA depletion abstract The main limiting factors for RNA-Seq analysis are quality and quantity of the isolated mRNA. In pro- karyotes, the proportion of messenger RNA to total RNA is rather low. Therefore, the main strategy of library preparation for sequencing is mRNA enrichment. Ribosomal and transfer RNAs, both mono- phosphorylated at the 5 0 -ends, are the major fractions of total RNA, while the bulk of primary transcripts is triphosphorylated at the 5 0 -teminus. Due to its low molecular weight, transfer RNA could be easily removed by a quick precipitation in LiCl solution. Ribosomal RNA may be degraded enzymatically by 5 0 - end terminal exonuclease XRN-1. These steps allow enriching samples in mRNA during the first stages of RNA-Seq library preparation. The desired level of fragmentation of enriched mRNA necessary for the 2 nd generation sequencing can be controlled by the duration of incubation at elevated temperatures in the presence of Mg 2þ -ions. Here, we describe a simple protocol for construction of the primary prokaryotic mRNA-saturated library without long depletion procedures. © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved. 1. Introduction RNA sequencing is a widespread application that requires a quality mRNA extract. However, the presence of large amounts of rRNA or tRNA in an isolated total RNA fraction creates certain issues for this type of analysis, especially in bacteria [1e4]. Prokaryotic RNA is phosphorylated at 5 0 end, and variability of this covalent modification allows distinguishing between different types of RNA in vitro. In general, 5 0 -triphosphorylation of Escherichia coli mRNA accounts for at least 15% of polysome-associated molecules [5], while 35e50% of mRNA exists in a 5 0 -diphosphate form [6]. The major part of mRNA degradation in the bacterial transcriptome goes through monophosphorylated intermediates, with the pri- mary 5 0 -tri- and 5 0 -diphosphorylated transcripts being converted into monophosphorylated counterparts by RNA 5 0 -pyrophospho- hydrolase (RppH) that triggers their subsequent degradation by RNAse E [7]. In turn, ribosomal RNA such as 16S, 23S and 5S and tRNAs is transcribed together as a 30S single triphosphorylated transcript. This precursor is then cleaved by RNAse III into three sub-precursors, each one then being processed by multiple RNAses that remove 5 0 - and 3 0 -end fragments to form mature rRNA mole- cules [8]. This processing is rapid, thus most rRNA is mature in E. coli. Only 1e2% of rRNA appears in a form of a single precursor, while 10e20% exist as the pre-precursors being cut by RNAse III [9]. Therefore, only the primary single transcript and the 16S precursor (prior to its 5 0 -terminal processing) are trisphosphorylated. Hence, the bulk of bacterial mature rRNA exists in a form of 5 0 -mono- phosphates, which accounts for more than 85% of total RNA content. Several strategies have been developed to remove rRNAs from total RNA. (1) Hybridization is a simple method which employs antisense rRNA probes to remove rRNA from total RNA samples. It allows reducing rRNA transcripts abundance by 40e60% [1 ,2]. However, the use of commercially available kits is often inconve- nient due to their limited applicability to a variety of non-model organisms. (2) RNAse-H based degradation of rRNA that uses syn- thetic single-stranded DNA oligonucleotides hybridized to rRNA * Corresponding author. E-mail addresses: ksmironov@ifr.moscow, ksmironov@ifr.moscow (K.S. Mironov). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi https://doi.org/10.1016/j.biochi.2020.08.001 0300-9084/© 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved. Biochimie 177 (2020) 63e67