Abstract
The ability to map transcription start sites is critical for studies of gene regulation and for identification of novel RNAs. Conventional RNA-seq is often insufficient for identification of transcription start sites due to low coverage and/or RNA processing events. We have developed a highly sensitive, genome-scale method for detection of transcription start sites in bacteria. This method uses deep sequencing of cDNA libraries to identify transcription start sites with strand specificity at nucleotide resolution. Here, we describe the application of this method for transcription start site identification in Escherichia coli.
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References
Waters LS, Storz G (2009) Regulatory RNAs in bacteria. Cell 136:615–628
Ozsolak F, Milos PM (2011) RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 12:87–98
Scotto-Lavino E, Du G, Frohman MA (2006) 5′ End cDNA amplification using classic RACE. Nat Protoc 1:2555–2562
Deana A, Celesnik H, Belasco JG (2008) The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal. Nature 451:355–358
Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, Chabas S, Reiche K, Hackermüller J, Reinhardt R, Stadler PF, Vogel J (2010) The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464:250–255
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:0008
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
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Singh, N., Wade, J.T. (2014). Identification of Regulatory RNA in Bacterial Genomes by Genome-Scale Mapping of Transcription Start Sites. In: Lafontaine, D., Dubé, A. (eds) Therapeutic Applications of Ribozymes and Riboswitches. Methods in Molecular Biology, vol 1103. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-730-3_1
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DOI: https://doi.org/10.1007/978-1-62703-730-3_1
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-729-7
Online ISBN: 978-1-62703-730-3
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