Skip to main content
SpringerLink
Log in

Analysis of Chromatin Accessibility, Histone Modifications, and Transcriptional States in Specific Cell Types Using Flow Cytometry

  • Protocol
  • First Online:
Plant Gene Regulatory Networks

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2698))

Abstract

The past two decades in biomedical research have experienced an explosion of cell type-specific and single-cell studies, especially concerning the concomitant dissection of regulatory and transcriptional landscapes of those under investigation. Additionally, leveraging next-generation sequencing (NGS) platforms efforts have been undertaken to evaluate the effects of chromatin accessibility, histone modifications, or even transcription factor binding sites. We have shown that Fluorescence-Activated Nuclear Sorting (FANS) is an effective means to characterize the transcriptomes of nuclei from different tissues. In light of our own technical and experimental developments, we extend this effort to combine FACS/FANS with Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) for profiling individual cell types according to their chromatin and transcriptional states.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 44.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 59.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815. https://doi.org/10.1038/35048692

    Article  Google Scholar 

  2. Birnbaum K, Shasha DE, Wang JY et al (2003) A gene expression map of the Arabidopsis root. Science 302:1956–1960. https://doi.org/10.1126/science.1090022

    Article  CAS  PubMed  Google Scholar 

  3. Brady SM, Orlando DA, Lee J-Y et al (2007) A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318:801–806. https://doi.org/10.1126/science.1146265

    Article  CAS  PubMed  Google Scholar 

  4. Yadav RK, Tavakkoli M, Xie M et al (2014) A high-resolution gene expression map of the Arabidopsis shoot meristem stem cell niche. Development 141:2735–2744. https://doi.org/10.1242/dev.106104

    Article  CAS  PubMed  Google Scholar 

  5. Slane D, Kong J, Berendzen KW et al (2014) Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo. Development 141:4831–4840. https://doi.org/10.1242/dev.116459

    Article  CAS  PubMed  Google Scholar 

  6. Adrian J, Chang J, Ballenger CE et al (2015) Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population. Dev Cell 33:107–118. https://doi.org/10.1016/j.devcel.2015.01.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dinneny JR, Long TA, Wang JY et al (2008) Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320:942–945. https://doi.org/10.1126/science.1153795

    Article  CAS  PubMed  Google Scholar 

  8. Contreras-López O, Vidal EA, Riveras E et al (2022) Spatiotemporal analysis identifies ABF2 and ABF3 as key hubs of endodermal response to nitrate. Proc Natl Acad Sci U S A 119:e2107879119. https://doi.org/10.1073/pnas.2107879119

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Weinhofer I, Hehenberger E, Roszak P et al (2010) H3K27me3 profiling of the endosperm implies exclusion of Polycomb group protein targeting by DNA methylation. PLoS Genet 6:e1001152. https://doi.org/10.1371/journal.pgen.1001152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lu Z, Hofmeister BT, Vollmers C et al (2017) Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes. Nucleic Acids Res 45:e41. https://doi.org/10.1093/nar/gkw1179

    Article  CAS  PubMed  Google Scholar 

  11. Lee LR, Wengier DL, Bergmann DC (2019) Cell-type-specific transcriptome and histone modification dynamics during cellular reprogramming in the Arabidopsis stomatal lineage. Proc Natl Acad Sci U S A 116:21914–21924. https://doi.org/10.1073/pnas.1911400116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Karaaslan ES, Faiß N, Liu C, Berendzen KW (2020) Isolation of lineage specific nuclei based on distinct endoreduplication levels and tissue-specific markers to study chromatin accessibility landscapes. Plan Theory 9:1478. https://doi.org/10.3390/plants9111478

    Article  CAS  Google Scholar 

  13. Denyer T, Ma X, Klesen S et al (2019) Spatiotemporal developmental trajectories in the Arabidopsis root revealed using high-throughput single-cell RNA sequencing. Dev Cell 48:840–852.e5. https://doi.org/10.1016/j.devcel.2019.02.022

    Article  CAS  PubMed  Google Scholar 

  14. Jean-Baptiste K, McFaline-Figueroa JL, Alexandre CM et al (2019) Dynamics of gene expression in single root cells of Arabidopsis thaliana. Plant Cell 31:993–1011. https://doi.org/10.1105/tpc.18.00785

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Farmer A, Thibivilliers S, Ryu KH et al (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. https://doi.org/10.1016/j.molp.2021.01.001

    Article  CAS  PubMed  Google Scholar 

  16. Lopez-Anido CB, Vatén A, Smoot NK et al (2021) Single-cell resolution of lineage trajectories in the Arabidopsis stomatal lineage and developing leaf. Dev Cell 56:1043–1055.e4. https://doi.org/10.1016/j.devcel.2021.03.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Dorrity MW, Alexandre CM, Hamm MO et al (2021) The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution. Nat Commun 12:3334. https://doi.org/10.1038/s41467-021-23675-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Slane D, Berendzen KW, Witthöft J, Jürgens G (2020) Transcriptomic profiling of the Arabidopsis embryonic epidermis using FANS in combination with RNAseq. Methods Mol Biol 2122:151–164. https://doi.org/10.1007/978-1-0716-0342-0_12

    Article  CAS  PubMed  Google Scholar 

  19. Galbraith DW, Sun G (2021) Flow cytometry and sorting in Arabidopsis. In: Sanchez-Serrano JJ, Salinas J (eds) Arabidopsis protocols. Springer US, New York, pp 255–294

    Chapter  Google Scholar 

  20. Gutzat R, Mittelsten Scheid O (2020) Preparing chromatin and RNA from rare cell types with Fluorescence-Activated Nuclear Sorting (FANS). In: Spillane C, McKeown P (eds) Plant epigenetics and epigenomics: methods and protocols. Springer US, New York, pp 95–105

    Chapter  Google Scholar 

  21. Loureiro J, Rodriguez E, Doležel J, Santos C (2007) Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100:875–888. https://doi.org/10.1093/aob/mcm152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Buenrostro JD, Wu B, Chang HY, Greenleaf WJ (2015) ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol 109:21.29.1–21.29.9. https://doi.org/10.1002/0471142727.mb2129s109

    Article  PubMed  PubMed Central  Google Scholar 

  23. Musielak TJ, Schenkel L, Kolb M et al (2015) A simple and versatile cell wall staining protocol to study plant reproduction. Plant Reprod 28:161–169. https://doi.org/10.1007/s00497-015-0267-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank the Deutsche Forschungsgemeinschaft (DFG Emmy Noether ProgP GR4251-1 to C.G.), the Japan Society for the Promotion of Science (JSPS), and the Tokyo University of Science Grant for International Joint Research (to T.S.) for funding. We thank Martin Bayer (ZMBP) for seed material and marker line images. We also thank Novogene and Macrogen for library sequencing and Bioanalyzer images.

Author information

Authors and Affiliations

  1. Center for Plant Molecular Biology, University of Tübingen, Tübingen, Germany

    Kenneth W. Berendzen

  2. Faculty of Biology and Biotechnology, Molecular and Cellular Botany, University of Bochum, Bochum, Germany

    Christopher Grefen

  3. Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba, Japan

    Takuya Sakamoto & Daniel Slane

  4. The University of Tokyo, Graduate School of Frontier Sciences, Department of Integrated Biosciences, Laboratory of Integrated Biology, Chiba, Japan

    Daniel Slane

Authors
  1. Kenneth W. Berendzen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher Grefen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takuya Sakamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel Slane
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel Slane .

Editor information

Editors and Affiliations

  1. Berlin, Germany

    Kerstin Kaufmann

  2. Ghent, Belgium

    Klaas Vandepoele

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Berendzen, K.W., Grefen, C., Sakamoto, T., Slane, D. (2023). Analysis of Chromatin Accessibility, Histone Modifications, and Transcriptional States in Specific Cell Types Using Flow Cytometry. In: Kaufmann, K., Vandepoele, K. (eds) Plant Gene Regulatory Networks. Methods in Molecular Biology, vol 2698. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3354-0_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3354-0_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3353-3

  • Online ISBN: 978-1-0716-3354-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation