Methods in Molecular Biology Volume 954, 2013, pp 555-566

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

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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.