Construction of prokaryotic strand-specific primary-transcripts saturated RNASeq library by controlled heat magnesium-dependent mRNA degradation

Kirill S. Mironov, Maria Shumskaya, Dmitry A. Los

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

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 2nd 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.

Original languageEnglish
Pages (from-to)63-67
Number of pages5
JournalBiochimie
Volume177
DOIs
StatePublished - Oct 2020

Keywords

  • cDNA library preparation
  • mRNA
  • Primary transcripts
  • RNASeq
  • rRNA depletion
  • XRN-1

Fingerprint

Dive into the research topics of 'Construction of prokaryotic strand-specific primary-transcripts saturated RNASeq library by controlled heat magnesium-dependent mRNA degradation'. Together they form a unique fingerprint.

Cite this