Abstract
Transcriptome profiling has been significantly hampered by the heterogeneity among individual cells within a tissue or an organ. Recent advances in single cell transcriptome profiling have significantly advanced our understanding of the transcriptome. However, plant single-cell RNA sequencing (scRNA-seq) relies on the isolation of protoplasts, which is not only impossible for many cell types but also induces acute wounding responses. To solve these problems, single-nucleus RNA sequencing (snRNA-seq) has been applied to plant research, in which nuclei are isolated and subject to encapsulation and profiling. Compared with scRNA-seq, snRNA-seq can be applied to a wider range of tissue types and plant species. Nevertheless, fewer transcripts can be obtained from each nucleus than each protoplast. In this chapter, we describe a detailed and general protocol to prepare nuclei from plant tissues that are ready for subsequent library construction and high-throughput sequencing.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
de Souza N (2010) Single-cell methods. Nat Methods 7:35
Mo Y, Jiao Y (2022) Advances and applications of single-cell omics technologies in plant research. Plant J 110:1551–1563
Bakken TE, Hodge RD, Miller JA, Yao ZZ, Nguyen TN, Aevermann B, Barkan E, Bertagnolli D, Casper T, Dee N, Garren E, Goldy J, Graybuck LT, Kroll M, Lasken RS, Lathia K, Parry S, Rimorin C, Scheuermann RH, Schork NJ, Shehata SI, Tieu M, Phillips JW, Bernard A, Smith KA, Zeng HK, Lein ES, Tasic B (2018) Single-nucleus and single-cell transcriptomes compared in matched cortical cell types. PLoS One 13:e0209648
Denisenko E, Guo BB, Jones M, Hou R, de Kock L, Lassmann T, Poppe D, Clement O, Simmons RK, Lister R, Forrest ARR (2020) Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows. Genome Biol 21:130
van den Brink SC, Sage F, Vertesy A, Spanjaard B, Peterson-Maduro J, Baron CS, Robin C, van Oudenaarden A (2017) Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat Methods 14:935–936
Wu HJ, Kirita Y, Donnelly EL, Humphreys BD (2019) Advantages of single-nucleus over single-cell RNA sequencing of adult kidney: rare cell types and novel cell states revealed in fibrosis. J Am Soc Nephrol 30:23–32
Farmer A, Thibivilliers S, Ryu KH, Schiefelbein J, Libault M (2021) Single-nucleus RNA and ATAC sequencing reveals the impact of chromatin accessibility on gene expression in Arabidopsis roots at the single-cell level. Mol Plant 14:372–383
Marand AP, Chen Z, Gallavotti A, Schmitz RJ (2021) A cis-regulatory atlas in maize at single-cell resolution. Cell 184:3041–3055.e21
Neumann M, Xu X, Smaczniak C, Schumacher J, Yan W, Bluthgen N, Greb T, Jonsson H, Traas J, Kaufmann K, Muino JM (2022) A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data. Nat Commun 13:2838
Tian C, Du Q, Xu M, Du F, Jiao Y (2020) Single-nucleus RNA-seq resolves spatiotemporal developmental trajectories in the tomato shoot apex. bioRxiv:2020.09.20.305029. https://doi.org/10.1101/2020.09.20.305029
Acknowledgments
This work was supported by the CAS Strategic Priority Research Program (grant XDA24020203) and the National Key R&D Program of China (grant 2023YFE0101100).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Xin, X., Du, F., Jiao, Y. (2023). Plant Nuclei Isolation for Single-Nucleus RNA Sequencing. In: Riechmann, J.L., Ferrándiz, C. (eds) Flower Development . Methods in Molecular Biology, vol 2686. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3299-4_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3299-4_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3298-7
Online ISBN: 978-1-0716-3299-4
eBook Packages: Springer Protocols