Serial Cryosectioning for the Spatial Transcriptomics of Plant Tissues
4 affiliations
- 1Biodiversity Research Center, Academia Sinica, Taipei, Taiwan
- 2Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
- 3Research Center for Information Technology Innovation, Academia Sinica, Taipei, Taiwan
- 4Department of Ecology and Evolution, University of Chicago, Chicago, IL, USA
Abstract
Spatial transcriptomics enables genome-wide gene expression profiling while preserving tissue architecture, making it a powerful approach for studying plant developmental transitions. However, preparing small and structurally complex plant tissues for spatial transcriptomics remains technically challenging because samples must be rapidly preserved, precisely oriented, serially sectioned, and accurately positioned within the limited capture area of the Visium slides. Here, we describe an optimized workflow for cryo-embedding, serial cryosectioning, section placement, and data analysis of small plant samples for 10x Genomics Visium spatial transcriptomics. Using maize seedling shoot apices as the target, this protocol includes preparation of custom molds for optimal cutting temperature embedding, rapid fresh sample embedding, serial cryosectioning, section-position marking for Visium HD workflows, and morphological quality assessment of replicate tissue slides before transcript capture. The associated data analysis workflow includes Space Ranger processing, Seurat-based normalization and Harmony integration, anatomical domain annotation, pseudobulk and developmental trend analyses, RNA velocity, pseudotime analysis, transcription factor network analysis, single-cell reference mapping, and 3D transcriptome reconstruction. This computational workflow was developed and tested using maize Visium V1 data, but not Visium HD data. This protocol was used to generate serial spatial transcriptomes of maize shoot apices and developing leaf primordia, enabling reconstruction of gene expression transitions from the shoot apical meristem to sequential leaf developmental stages. The approach is also applicable to other small plant tissues, including Arabidopsis first true leaves and Marchantia thalli.
Key features
• Optimized cryo-embedding and cryosectioning workflow for small plant tissues for 10x Visium spatial transcriptomics.
• A streamlined computational workflow for serial-section processing, spatial-domain annotation, 3D reconstruction, and developmental trajectory analysis.
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BibTeX
@article{wu2026serial,
title = {{Serial Cryosectioning for the Spatial Transcriptomics of Plant Tissues}},
author = {Chi-Chih Wu and Kun-Ting Hsieh and Chun-Ping Yu and Yi-Hua Chen and Shu-Jen Chou and Ting-Ying Wu and Chin-Min Kimmy Ho and Shu-Hsing Wu and Mei-Yeh Jade Lu and Wen-Hsiung Li},
journal = {Bio-protocol},
year = {2026},
month = oct,
doi = {10.21769/bioprotoc.5844},
url = {https://doi.org/10.21769/bioprotoc.5844},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Serial Cryosectioning for the Spatial Transcriptomics of Plant Tissues
AU - Chi-Chih Wu
AU - Kun-Ting Hsieh
AU - Chun-Ping Yu
AU - Yi-Hua Chen
AU - Shu-Jen Chou
AU - Ting-Ying Wu
AU - Chin-Min Kimmy Ho
AU - Shu-Hsing Wu
AU - Mei-Yeh Jade Lu
AU - Wen-Hsiung Li
PY - 2026
DA - 2026/10/20
JO - Bio-protocol
DO - 10.21769/bioprotoc.5844
UR - https://doi.org/10.21769/bioprotoc.5844
AB - Spatial transcriptomics enables genome-wide gene expression profiling while preserving tissue architecture, making it a powerful approach for studying plant developmental transitions. However, preparing small and structurally complex plant tissues for spatial transcriptomics remains technically challenging because samples must be rapidly preserved, precisely oriented, serially sectioned, and accurately positioned within the limited capture area of the Visium slides. Here, we describe an optimized workflow for cryo-embedding, serial cryosectioning, section placement, and data analysis of small plant samples for 10x Genomics Visium spatial transcriptomics. Using maize seedling shoot apices as the target, this protocol includes preparation of custom molds for optimal cutting temperature embedding, rapid fresh sample embedding, serial cryosectioning, section-position marking for Visium HD workflows, and morphological quality assessment of replicate tissue slides before transcript capture. The associated data analysis workflow includes Space Ranger processing, Seurat-based normalization and Harmony integration, anatomical domain annotation, pseudobulk and developmental trend analyses, RNA velocity, pseudotime analysis, transcription factor network analysis, single-cell reference mapping, and 3D transcriptome reconstruction. This computational workflow was developed and tested using maize Visium V1 data, but not Visium HD data. This protocol was used to generate serial spatial transcriptomes of maize shoot apices and developing leaf primordia, enabling reconstruction of gene expression transitions from the shoot apical meristem to sequential leaf developmental stages. The approach is also applicable to other small plant tissues, including Arabidopsis first true leaves and Marchantia thalli. Key features • Optimized cryo-embedding and cryosectioning workflow for small plant tissues for 10x Visium spatial transcriptomics. • A streamlined computational workflow for serial-section processing, spatial-domain annotation, 3D reconstruction, and developmental trajectory analysis.
SN - 2331-8325
ER -