Methods in Molecular Biology Volume 954, 2013, pp 555-566
Date: 17 Oct 2012

cDNA Library Construction for Next-Generation Sequencing to Determine the Transcriptional Landscape of Legionella pneumophila

Download Book (9,321 KB) As a courtesy to our readers the eBook is provided DRM-free. However, please note that Springer uses effective methods and state-of-the art technology to detect, stop, and prosecute illegal sharing to safeguard our authors’ interests.
Download Protocol (297 KB)

Abstract

The adaptation of Legionella pneumophila to the different conditions it encounters in the environment and in the host is governed by a complex regulatory system. Current knowledge of these regulatory networks and the transcriptome responses of L. pneumophila is mainly based on microarray analysis and limited to transcriptional products of annotated protein-coding genes. The application of the Next-Generation Sequencing (NGS) technology allows now genome-wide strand-specific sequencing and accurate determination of all expressed regions of the genome to reveal the complete transcriptional network and the dynamic interplay of specific regulators on a genome-wide level. NGS-based techniques promote deeper understanding of the global transcriptional organization of L. pneumophila by identifying transcription start sites (TSS), alternative TSS and operon organization, noncoding RNAs, antisense RNAs, and 5′-/3′-untranslated regions. In this chapter we describe the construction of cDNA libraries for (1) RNA deep sequencing (RNA-seq) and (2) TSS mapping using the Illumina technology.