Abstract
Whole genome wide analysis of transcription using RNA-Seq methods is a powerful way to elucidate differential expression of gene features in bacteria across different conditions as well as for discovering previously exotic RNA species. Indeed, RNA sequencing has revolutionized the study of bacterial transcription with the diversity and quantity of small noncoding RNA elements that have been found and its ability to clearly define operons, promoters , and terminators . We discuss our experience with applying RNA sequencing technology to analyzing the lytic cycle, including extraction, processing, and a guide to the customized statistical analysis necessary for analyzing differential host and phage transcription.
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References
Oshlack A, Robinson MD, Young MD (2010) From RNA-Seq reads to differential expression results. Genome Biol 11:220. doi:10.1186/gb-2010-11-12-220
Ceyssens P, Minakhin L, Van den Bossche A, Yakunina M, Klimuk E, Blasdel B, De Smet J, Noben J, Bläsi U, Severinov K, Lavigne R (2014) Development of giant bacteriophage ФKZ is independent of the host transcription apparatus. J Virol 88(18):10501–10510
Wagemans J, Blasdel B, Van den Bossche A, Uytterhoeven B, De Smet J, Paeshuyse J, Cenens W, Aertsen A, Uetz P, Delattre A, Ceyssens P, Lavigne R (2014) Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa. Cell Microbiol 16(12):1822–1835
Georg J, Hess WR (2011) cis-antisense RNA, another level of gene regulation in bacteria. Microbiol Mol Biol Rev 75(2):286–300
Mills JD, Kawahara Y, Janitz M (2013) Strand-specific RNA-Seq provides greater resolution of transcriptome profiling. Curr Genomics 14(3):173–181
Croucher NJ, Fookes MC, Perkins TT, Turner DJ, Marguerat SB, Keane T, Quail MA, He M, Assefa S, Bähler J, Kingsley RA, Parkhill J, Bentley SD, Dougan G, Thomson NR (2009) A simple method for directional transcriptome sequencing using Illumina technology. Nucleic Acids Res 37(22):e148
Zhang Z, Theurkauf WE, Weng Z, Zamore PD (2012) Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection. Silence 3:9. doi:10.1186/1758-907X-3-9
Levin JZ, Yassour M, Adiconis X, Nusbaum C, Thompson DA, Friedman N, Gnirke A, Regev A (2010) Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat Methods 7(9):709–715
Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinformatics 1(5):589–595
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-Seq experiments with TopHat and Cufflinks. Nat Protoc 1(3):562–578
Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:10. doi:10.1186/gb-2010-11-10-r106
Rountree PM (1951) The role of certain electrolytes in the adsorption of staphylococcal bacteriophages. Microbiology 5(4):673–680
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Blasdel, B., Ceyssens, PJ., Lavigne, R. (2018). Preparing cDNA Libraries from Lytic Phage-Infected Cells for Whole Transcriptome Analysis by RNA-Seq. In: Clokie, M., Kropinski, A., Lavigne, R. (eds) Bacteriophages. Methods in Molecular Biology, vol 1681. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7343-9_14
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DOI: https://doi.org/10.1007/978-1-4939-7343-9_14
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