Abstract
RNA-sequencing (RNA-seq) provides invaluable knowledge on developmental pathways and the effects of mutant phenotypes. Plant reproductive cells have traditionally been difficult to isolate for genomics because they are rare and often deeply embedded within somatic tissues. Here, we present a protocol to isolate single maize meiocytes and pollen grains for RNA-seq. We discuss how to identify and isolate each sample type under a microscope, prepare RNA-seq libraries and analyze the data. This technique has several advantages over alternative methods, combining the ability to target specific rare cell types while resolving cell-to-cell heterogeneity with single-cell RNA-seq. The technique is compatible with minute amounts of starting material (e.g., a single anther), making it possible to collect dense time courses. Furthermore, developmentally synchronized anthers are saved for microscopy, allowing staging to be performed in parallel with expression analysis. Up to 200 cells can be collected in 4–5 h by someone proficient in tissue dissection, and library preparation can be completed in 2 d by researchers experienced in molecular biology and genomics. This protocol will facilitate research on plant reproduction, providing insights critical to plant breeding, genetics and agriculture.
Key points
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In this protocol, maize meiocytes and pollen grains are microscopically identified and isolated for single-cell RNA-seq by using a modified CEL-Seq2 workflow. Developmental staging of anthers is also performed in parallel with expression analysis.
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Rare reproductive cells are specifically targeted, and larger cell sizes and sample numbers are accommodated than in droplet-based single-cell RNA-seq methods. This protocol is compatible with minute amounts of material and resolves heterogeneity by focusing on single cells and pollen grains.
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Data availability
The example sequencing data analyzed in this protocol are available on the Gene Expression Omnibus under accession GSE121039. The data in Fig. 5 were reanalyzed from refs. 3,4 (Gene Expression Omnibus accessions GSE121039 and GSE175505). The ‘data analysis’ section can be followed by using example data from ref. 3, which can be accessed on the Sequence Read Archive under accessions SRR7989735 and SRR7989736.
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Acknowledgements
The authors thank V. Walbot, who provided invaluable guidance during the development of this technique, and J. Gent for his helpful feedback and suggestions. This work was supported by NSF grant no. 2218712 to B.N. J.A.-F. was supported by CONACyT PhD fellowship 781792 and by CONACyT Ciencia Básica 2017-2018 grant A1-S-34956.
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Nature Protocols thanks Chloé Girard, Marc Libault and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Related links
Key references using this protocol:
Nelms, B. & Walbot, V. Science 364, 52–56 (2019): https://doi.org/10.1126/science.aav6428
Nelms, B. & Walbot, V. Science 375, 424–429 (2022): https://doi.org/10.1126/science.abl7392
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2 and Supplementary Table 2
Supplementary Table
Supplementary Table 1. Sequence of barcoded CEL-seq primers. The first of these primers is shown in Supplementary Figure 2a with each section annotated. This table can be directly uploaded to IDT for ordering plate-based oligos. Because there are randomized bases in these primers (Ns), the automated form will ask if you want ‘machine-mixed’ or ‘hand-mixed’ bases during ordering; select machine-mixed bases.
Supplementary Data 1
Supplementary Data 1. CELseq_whitelist_legacy.txt. Legacy CELseq whitelist for Salmon Alevin. Use when re-analyzing data from ref. 3.
Supplementary Data 2
Supplementary Data 2. TGMAP.tsv. Transcript to gene mapping for Salmon Alevin.
Supplementary Data 3
Supplementary Data 3. CELseq_whitelist.txt. CELseq whitelist for Salmon Alevin. Use when analyzing new data generated with this protocol.
Supplementary Video 1
Supplementary Video 1. Technique to press anthers and release meiotic cells or pollen precursors (Step 5A(v–vii) and 5B(ii)).
Supplementary Video 2
Supplementary Video 2. Technique to isolate single cells and pollen precursors by using a syringe needle (Steps 6–12).
Supplementary Video 3
Supplementary Video 3. Handling of single-cell material on PCR caps (Steps 14 and 22–25).
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Washburn, M., Alaniz-Fabián, J., Scroggs, T. et al. Single-cell RNA-seq of maize meiocytes and pollen grains. Nat Protoc 18, 3512–3533 (2023). https://doi.org/10.1038/s41596-023-00889-6
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DOI: https://doi.org/10.1038/s41596-023-00889-6
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