Preparation of plant tissue to enable Spatial Transcriptomics profiling using barcoded microarrays

Abstract

Elucidation of the complex processes involved in plant growth requires analysis of the spatial gene expression patterns in all affected tissues. This protocol extension is an adaptation of a protocol that describes how to use barcoded oligo-dT microarrays to evaluate spatial global gene expression profiles in mammalian tissue to enable it to be applied to plant material. Here, we explain the required adjustments for preparing and treating plant tissue sections on the array surface, specifically in regard to how to permeabilize and remove the tissue. Once the tissue has been removed, the cDNA–mRNA hybrid that is left on the slide is processed in the same way as cDNA obtained during experiments on mammalian tissue; thus the later stages of the protocol are not included here, and readers should follow the accompanying protocol for those. We have previously used our protocol to generate high-quality sequencing libraries for Arabidopsis thaliana inflorescence, Populus tremula developing and dormant leaf buds, and Picea abies female cones. However, we anticipate that the protocol can be adapted to other tissue types and species. The entire protocol for preparing samples and processing libraries can be completed in 3–4 d.

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Fig. 1: Complete workflow for tissue-optimization experiments and generation of Spatial Transcriptomics sequencing libraries.
Fig. 2: Barcoded oligo-dT microarray resolution.
Fig. 3: Successful and unsuccessful tissue removal.
Fig. 4: Analysis of secondary metabolites.
Fig. 5: cDNA footprint.

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Acknowledgements

This work was supported by the Knut and Alice Wallenberg Foundation and the Swedish Research Council.

Author information

S.G. and J.L. designed the research. S.G. developed the protocol and wrote the manuscript.

Correspondence to Stefania Giacomello or Joakim Lundeberg.

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Competing interests

J.L. is the founder of a company that holds IP rights to the presented technology. S.G. declares no competing interests.

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Key reference using this protocol

Giacomello, S. et al. Nat. Plants 3, 17061 (2017): https://doi.org/10.1038/nplants.2017.61

This protocol is an extension to: Nat. Protoc. https://doi.org/10.1038/s41596-018-0045-2

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Giacomello, S., Lundeberg, J. Preparation of plant tissue to enable Spatial Transcriptomics profiling using barcoded microarrays. Nat Protoc 13, 2425–2446 (2018). https://doi.org/10.1038/s41596-018-0046-1

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