cDNA library construction for next-generation sequencing to determine the transcriptional landscape of Legionella pneumophila

Tobias Sahr (1) , Carmen Buchrieser (1)

1 Biologie des bactéries intracellulaires

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.

Mots clés: Legionella pneumophila; Next-generation sequencing; Transcriptional start site mapping; Small ncRNA

Domaines: Biologie moléculaire

Tobias Sahr, Carmen Buchrieser. cDNA library construction for next-generation sequencing to determine the transcriptional landscape of Legionella pneumophila. Carmen Buchrieser; Hubert Hilbi. Legionella: Methods and Protocols, 954, Humana Press, pp.555-566, 2013, Methods in Molecular Biology (MIMB), 978-1-62703-160-8 (Hardcover), 978-1-4939-6274-7 (Softcover), 978-1-62703-161-5 (eBook). ⟨10.1007/978-1-62703-161-5_34⟩. ⟨pasteur-01334075⟩