Abstract
Identifying the transcription start sites (TSS) of genes is essential for characterizing promoter regions. Several protocols have been developed to capture the 5′ end of transcripts via Cap-Analysis of Gene Expression (CAGE) or linker-ligation strategies such as Paired-End Analysis of Transcription Start Sites (PEAT), but often require large amounts of tissue. More recently, nanoCAGE was developed for sequencing on the Illumina GAIIx to overcome this limitation. In this chapter, we present the nanoCAGE-XL protocol, the first publicly available adaptation of nanoCAGE for sequencing on recent ultra-high-throughput platforms such as Illumina HiSeq-2000. NanoCAGE-XL provides a method for precise transcription start site identification in large eukaryotic genomes, even in cases where input total RNA quantity is very limited.
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References
Plessy C, Bertin N, Takahashi H, Simone R, Salimullah M, Lassmann T, Vitezic M, Severin J, Olivarius S, Lazarevic D, Hornig N, Orlando V, Bell I, Gao H, Dumais J, Kapranov P, Wang H, Davis CA, Gingeras TR, Kawai J, Daub CO, Hayashizaki Y, Gustincich S, Carninci P (2010) Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan. Nat Methods 7:528–534
Megraw M, Pereira F, Jensen ST, Ohler U, Hatzigeorgiou AG (2009) A transcription factor affinity-based code for mammalian transcription initiation. Genome Res 19:644–656
Morton T, Petricka J, Corcoran DL, Li S, Winter CM, Carda A, Benfey PN, Ohler U, Megraw M (2014) Paired-end analysis of transcription start sites in Arabidopsis reveals plant-specific promoter signatures. Plant Cell 26:2746–2760
Ni T, Corcoran DL, Rach EA, Song S, Spana EP, Gao Y, Ohler U, Zhu J (2010) A paired-end sequencing strategy to map the complex landscape of transcription initiation. Nat Methods 7:521–527
Takahashi H, Kato S, Murata M, Carninci P (2012) CAGE (cap analysis of gene expression): a protocol for the detection of promoter and transcriptional networks. Methods Mol Biol 786:181–200
Batut P, Dobin A, Plessy C, Carninci P, Gingeras TR (2013) High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression. Genome Res 23:169–180
Nepal C, Hadzhiev Y, Previti C, Haberle V, Li N, Takahashi H, Suzuki AM, Sheng Y, Abdelhamid RF, Anand S, Gehrig J, Akalin A, Kockx CE, van der Sloot AA, van Ijcken WF, Armant O, Rastegar S, Watson C, Strahle U, Stupka E, Carninci P, Lenhard B, Muller F (2013) Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis. Genome Res 23:1938–1950
Salimullah M, Sakai M, Plessy C, Carninci P (2011, 2011) NanoCAGE: a high-resolution technique to discover and interrogate cell transcriptomes. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot5559 pdb prot5559
Tang DT, Plessy C, Salimullah M, Suzuki AM, Calligaris R, Gustincich S, Carninci P (2012) Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching. Nucleic Acids Res 41:e44
Batut P, Gingeras TR (2013) RAMPAGE: promoter activity profiling by paired-end sequencing of 5'-complete cDNAs. Curr Protoc Mol Biol 104:Unit 25B 11. doi:https://doi.org/10.1002/0471142727.mb25b11s104
Marques AC, Hughes J, Graham B, Kowalczyk MS, Higgs DR, Ponting CP (2013) Chromatin signatures at transcriptional start sites separate two equally populated yet distinct classes of intergenic long noncoding RNAs. Genome Biol 14:R131
Cumbie JS, Ivanchenko MG, Megraw M (2015) NanoCAGE-XL and CapFilter: an approach to genome wide identification of high confidence transcription start sites. BMC Genomics 16:597. https://doi.org/10.1186/s12864-015-1670-6
Acknowledgments
We would like to thank Charles Plessy of the RIKEN Center for Life Science Technologies and Jenn To of Grassroots Biotechnology for technical advice on the nanoCAGE protocol. We would also like to thank Mark Dasenko of the Center for Genome Research and Biocomputing at Oregon State University for troubleshooting assistance in sample preparation for sequencing. This work was supported by NIH grant GM097188 and startup funds from Oregon State University to M.M.
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Ivanchenko, M.G., Megraw, M. (2018). NanoCAGE-XL: An Approach to High-Confidence Transcription Start Site Sequencing. In: Yamaguchi, N. (eds) Plant Transcription Factors. Methods in Molecular Biology, vol 1830. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8657-6_13
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DOI: https://doi.org/10.1007/978-1-4939-8657-6_13
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