Abstract
Degradome sequencing provides large amounts of data regarding RNA degradation. The degradome library construction described here is modified from the 5′-rapid amplification of cDNA ends (5′-RACE), and each degradome cDNA is sequenced by next-generation sequencing (NGS). Degradome profiles provide information confirming miRNA-mediated cleavage of target genes and allow the identification of novel targets. Furthermore, degradome sequencing provides additional information for the study of RNA processing, such as information regarding RNA-binding proteins. In this chapter, we describe a detailed optimized protocol for constructing a degradome library with high yield and quality, along with NGS and data mining procedures. We hope that the degradome approach will be applied to further studies of non-model organisms.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Souret FF, Kastenmayer JP, Green PJ (2004) AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets. Mol Cell 15:173–183
Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24:1420–1428
Zhai J, Arikit S, Simon SA, Kingham BF, Meyers BC (2014) Rapid construction of parallel analysis of RNA end (PARE) libraries for Illumina sequencing. Methods 67:84–90
Lin PC, Lu CW, Shen BN, Lee GZ, Bowman JL, Arteaga-Vazquez MA, Liu LY, Hong SF, Lo CF, Su GM, Kohchi T, Ishizaki K, Zachgo S, Althoff F, Takenaka M, Yamato KT, Lin SS (2016) Identification of miRNAs and their targets in the liverwort Marchantia polymorpha by integrating RNA-Seq and degradome analyses. Plant Cell Physiol 57:339–358
Hou CY, Wu MT, Lu SH, Hsing YI, Chen HM (2014) Beyond cleaved small RNA targets: unraveling the complexity of plant RNA degradome data. BMC Genomics 15:15
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120
Acknowledgment
We are grateful to the High Throughput Genomics Core at the Biodiversity Research Center, Academia Sinica, for performing Illumina sequencing. The study described in this chapter was supported by a grant from the Ministry of Science and Technology of Taiwan under contract number MOST 106-2321-B-002-008.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Lin, SS., Chen, Y., Lu, MY.J. (2019). Degradome Sequencing in Plants. In: de Folter, S. (eds) Plant MicroRNAs. Methods in Molecular Biology, vol 1932. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9042-9_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9042-9_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9041-2
Online ISBN: 978-1-4939-9042-9
eBook Packages: Springer Protocols