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Plant Transcription Factors pp 191–224Cite as

Laser Capture Micro-Dissection Coupled to RNA Sequencing: A Powerful Approach Applied to the Model Legume Medicago truncatula in Interaction with Sinorhizobium meliloti

Part of the Methods in Molecular Biology book series (MIMB,volume 1830)

Abstract

Understanding the development of multicellular organisms requires the identification of regulators, notably transcription factors, and specific transcript populations associated with tissue differentiation. Laser capture microdissection (LCM) is one of the techniques that enable the analysis of distinct tissues or cells within an organ. Coupling this technique with RNA sequencing (RNAseq) makes it extremely powerful to obtain a genome-wide and dynamic view of gene expression. Moreover, RNA sequencing allows two or potentially more interacting organisms to be analyzed simultaneously. In this chapter, a LCM-RNAseq protocol optimized for root and symbiotic root nodule analysis is presented, using the model legume Medicago truncatula (in interaction with Sinorhizobium meliloti in the nodule samples). This includes the description of procedures for plant material fixation, embedding, and micro-dissection; it is followed by a presentation of techniques for RNA extraction and amplification, adapted for the simultaneous analysis of plant and bacterial cells in interaction or, more generally, polyadenylated and non-polyadenylated RNAs. Finally, step-by-step statistical analyses of RNAseq data are described. Those are critical for quality assessment of the whole procedure and for the identification of differentially expressed genes.

Key words

  • Laser capture micro-dissection
  • RNA sequencing
  • rRNA depletion
  • Root
  • Nodule
  • Statistical analyses

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Acknowledgments

This work was supported by a grant from the Agence Nationale de la Recherche (ANR) (SYMbiMICS project). We are grateful to Jérôme Gouzy (LIPM, Toulouse) who contributed to launch the SYMbiMICS project and provided strong bioinformatics support. We thank Yves Martinez (imagery platform, FR AIB, Toulouse) for his advice on the LCM equipment, Olivier Catrice (LIPM) and Antonius Timmers (LIPM) for their advice on plant sample fixing and embedding, as well as Yann Pecrix (LIPM) and Marc Ellis for critical reading of this manuscript. The microdissection equipment was provided by the FR AIB imagery platform.

Author information

Author notes

  1. Brice Roux

    Present address: BIAM, Université Aix-Marseille, CNRS, CEA, Saint-Paul-lez-Durance, France

  2. Nathalie Rodde

    Present address: CNRGV, INRA, Castanet-Tolosan, France

Authors and Affiliations

  1. LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France

    Brice Roux, Nathalie Rodde, Sandra Moreau, Marie-Françoise Jardinaud & Pascal Gamas

  2. INPT-Université de Toulouse, ENSAT, Castanet-Tolosan, France

    Marie-Françoise Jardinaud

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  1. Brice Roux
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  2. Nathalie Rodde
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  3. Sandra Moreau
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  4. Marie-Françoise Jardinaud
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  5. Pascal Gamas
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Corresponding author

Correspondence to Pascal Gamas .

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Editors and Affiliations

  1. Plant Stem Cell Regulation and Floral Patterning Laboratory, Nara Institute of Science and Technology, Ikoma, Nara, Japan

    Nobutoshi Yamaguchi

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Roux, B., Rodde, N., Moreau, S., Jardinaud, MF., Gamas, P. (2018). Laser Capture Micro-Dissection Coupled to RNA Sequencing: A Powerful Approach Applied to the Model Legume Medicago truncatula in Interaction with Sinorhizobium meliloti . In: Yamaguchi, N. (eds) Plant Transcription Factors. Methods in Molecular Biology, vol 1830. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8657-6_12

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  • DOI: https://doi.org/10.1007/978-1-4939-8657-6_12

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