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Preparing Chromatin and RNA from Rare Cell Types with Fluorescence-Activated Nuclear Sorting (FANS)

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Plant Epigenetics and Epigenomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2093))

Abstract

The application of fluorescent tags to generate cell type-specific translational and transcriptional reporter lines is routine in plants, but separation of different cell types for downstream analyses is hampered by the presence of cell walls and tight connections between cells. Enzymatic removal of cell walls induces a wound response, dedifferentiation, or reprogramming of the resulting protoplasts. Their osmotic and mechanical instability and their large size range are challenging for FACS, a flow -sorting procedure based on differential expression of fluorescent tags. In contrast, plant nuclei are relatively robust and easy to isolate. Here, we describe a protocol for fluorescence-activated nuclear sorting (FANS) that allows efficient purification of very few fluorescence-tagged nuclei from a large background of non-labeled tissue. Purified nuclei are suitable for genome, epigenome, transcriptome, or proteome analyses. We describe in detail how to analyze nuclear RNA and DNA methylation from sorted nuclei representing the limited number of stem cells in the shoot apical meristem of Arabidopsis.

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References

  1. Gutzat R, Mittelsten Scheid O (2012) Epigenetic responses to stress: triple defense? Curr Opin Plant Biol 15(5):568–573. https://doi.org/10.1016/j.pbi.2012.08.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Vriet C, Hennig L, Laloi C (2015) Stress-induced chromatin changes in plants: of memories, metabolites and crop improvement. Cell Mol Life Sci 72(7):1261–1273

    Article  CAS  PubMed  Google Scholar 

  3. Borg M, Berger F (2015) Chromatin remodelling during male gametophyte development. Plant J 83(1):177–188. https://doi.org/10.1111/tpj.12856

    Article  CAS  PubMed  Google Scholar 

  4. Pecinka A, Dinh HQ, Baubec T, Rosa M, Lettner N, Mittelsten Scheid O (2010) Epigenetic regulation of repetitive elements is attenuated by prolonged heat stress in Arabidopsis. Plant Cell 22(9):3118–3129. https://doi.org/10.1105/tpc.110.078493

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Feng XQ, Zilberman D, Dickinson H (2013) A conversation across generations: soma-germ cell crosstalk in plants. Dev Cell 24(3):215–225. https://doi.org/10.1016/j.devcel.2013.01.014

    Article  CAS  PubMed  Google Scholar 

  6. Gutzat R, Rembart K, Nussbaumer T, Pisupati R, Hofmann F, Bradamant G, Daubel N, Gaidora A, Lettner N, Donà M, Nordborg M, Nodine M, Mittelsten Scheid O (2018) Stage-specific transcriptomes and DNA methylomes indicate an early and transient loss of transposon control in Arabidopsis shoot stem cells. bioRxiv:430447. https://doi.org/10.1101/430447

  7. Kawakatsu T, Stuart T, Valdes M, Breakfield N, Schmitz RJ, Nery JR, Urich MA, Han XW, Lister R, Benfey PN, Ecker JR (2016) Unique cell-type-specific patterns of DNA methylation in the root meristem. Nat Plants 2(5):16058. https://doi.org/10.1038/nplants.2016.58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kawakatsu T, Huang SSC, Jupe F, Sasaki E, Schmitz RJ, Urich MA, Castanon R, Nery JR, Barragan C, He YP, Chen HM, Dubin M, Lee CR, Wang CM, Bemm F, Becker C, O’Neil R, O’Malley RC, Quarless DX, Schork NJ, Weigel D, Nordborg M, Ecker JR, Genomes C (2016) Epigenomic diversity in a global collection of Arabidopsis thaliana accessions. Cell 166(2):492–505. https://doi.org/10.1016/j.cell.2016.06.044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dixit R, Cyr R, Gilroy S (2006) Using intrinsically fluorescent proteins for plant cell imaging. Plant J 45(4):599–615. https://doi.org/10.1111/j.1365-313X.2006.02658.x

    Article  CAS  PubMed  Google Scholar 

  10. Tanz SK, Castleden I, Small ID, Millar AH (2013) Fluorescent protein tagging as a tool to define the subcellular distribution of proteins in plants. Front Plant Sci 4:214. https://doi.org/10.3389/fpls.2013.00214

    Article  PubMed  PubMed Central  Google Scholar 

  11. Carter AD, Bonyadi R, Gifford ML (2013) The use of fluorescence-activated cell sorting in studying plant development and environmental responses. Int J Dev Biol 57(6–8):545–552. https://doi.org/10.1387/ijdb.130195mg

    Article  CAS  PubMed  Google Scholar 

  12. Deal RB, Henikoff S (2011) The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc 6(1):56–68

    Article  CAS  PubMed  Google Scholar 

  13. Sijacic P, Bajic M, McKinney EC, Meagher RB, Deal RB (2018) Changes in chromatin accessibility between Arabidopsis stem cells and mesophyll cells illuminate cell type-specific transcription factor networks. Plant J 94(2):215–231. https://doi.org/10.1111/tpj.13882

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. You Y, Sawikowska A, Neumann M, Pose D, Capovilla G, Langenecker T, Neher RA, Krajewski P, Schmid M (2017) Temporal dynamics of gene expression and histone marks at the Arabidopsis shoot meristem during flowering. Nat Commun 8:15120. https://doi.org/10.1038/ncomms15120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhang C, Barthelson RA, Lambert GM, Galbraith DW (2008) Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiol 147(1):30–40. https://doi.org/10.1104/pp.107.115246

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Weinhofer I, Köhler C (2014) Endosperm-specific chromatin profiling by fluorescence-activated nuclei sorting and chip-on-chip. Methods Mol Biol 1112:105–115

    Article  CAS  PubMed  Google Scholar 

  17. Lu ZF, Hofmeister BT, Vollmers C, DuBois RM, Schmitz RJ (2017) Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes. Nucleic Acids Res 45(6):e41. https://doi.org/10.1093/nar/gkw1179

    Article  CAS  PubMed  Google Scholar 

  18. Slane D, Bayer M (2017) Cell type-specific gene expression profiling using fluorescence-activated nuclear sorting. Methods Mol Biol 1629:27–35

    Article  CAS  PubMed  Google Scholar 

  19. Pavlova P, Tessadori F, de Jong HJ, Fransz P (2010) Immunocytological analysis of chromatin in isolated nuclei. Methods Mol Biol 655:413–432. https://doi.org/10.1007/978-1-60761-765-5_28

    Article  CAS  PubMed  Google Scholar 

  20. Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220(4601):1049–1051

    Article  CAS  PubMed  Google Scholar 

  21. Picelli S, Faridani OR, Bjorklund AK, Winberg G, Sagasser S, Sandberg R (2014) Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9(1):171–181. https://doi.org/10.1038/nprot.2014.006

    Article  CAS  PubMed  Google Scholar 

  22. Dolezel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2(9):2233–2244. https://doi.org/10.1038/nprot.2007.310

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, Austria

    Ruben Gutzat & Ortrun Mittelsten Scheid

Authors
  1. Ruben Gutzat
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  2. Ortrun Mittelsten Scheid
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Corresponding author

Correspondence to Ortrun Mittelsten Scheid .

Editor information

Editors and Affiliations

  1. Plant and AgriBiosciences Research Centre, Ryan Institute, National University of Ireland Galway (NUI Galway), Galway, Ireland

    Charles Spillane

  2. Plant and AgriBiosciences Research Centre, Ryan Institute, National University of Ireland Galway (NUI Galway), Galway, Ireland

    Peter McKeown

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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 in Molecular Biology, vol 2093. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0179-2_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0179-2_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0178-5

  • Online ISBN: 978-1-0716-0179-2

  • eBook Packages: Springer Protocols

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