The main limiting factors for RNA-Seq analysis are quality and quantity of the isolated mRNA. In prokaryotes, 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 monophosphorylated at the 5′-ends, are the major fractions of total RNA, while the bulk of primary transcripts is triphosphorylated at the 5′-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′-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 Mg²⁺-ions. Here, we describe a simple protocol for construction of the primary prokaryotic mRNA-saturated library without long depletion procedures.

RNA-Seq分析的主要限制因素是分离的mRNA的质量和数量。在原核生物中，信使RNA与总RNA的比例相当低。因此，用于测序的文库制备的主要策略是mRNA富集。核糖体和转移RNA均在5'端单磷酸化，是总RNA的主要部分，而大部分初级转录本在5'端三磷酸化。由于其分子量低，可以通过在LiCl溶液中快速沉淀来轻松去除转移RNA。核糖体RNA可以被5'端末端核酸外切酶XRN-1酶促降解。这些步骤允许在RNA-Seq文库制备的最初阶段富集mRNA中的样品。所必需的2富集的mRNA的片段化的期望电平^第二世代测序可以通过在Mg ^2+离子存在下在高温下孵育的持续时间来控制。在这里，我们描述了一个简单的协议，用于构建原核生物mRNA饱和的文库，而无需花费很长时间。