title: cDNA library construction for next-generation sequencing to determine the transcriptional landscape of Legionella pneumophila.
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.
authors.affiliation: Biologie des Bactéries Intracellulaires, Institut Pasteur, Paris, France.
authors.forename: Tobias
authors.initials: T
authors.lastname: Sahr
authors.forename: Carmen
authors.initials: C
authors.lastname: Buchrieser
mesh_terms: D020869:Gene Expression Profiling; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D016952:Legionella pneumophila / Q000235:genetics*; D012329:RNA, Bacterial / Q000302:isolation & purification; D024363:Transcription Initiation Site; D059467:Transcriptome
chemical_list: D012329:RNA, Bacterial