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  • Protocol Extension
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Applying the INTACT method to purify endosperm nuclei and to generate parental-specific epigenome profiles

Nature Protocols volume 12, pages 238–254 (2017)Cite this article

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Abstract

The early endosperm tissue of dicot species is very difficult to isolate by manual dissection. This protocol details how to apply the INTACT (isolation of nuclei tagged in specific cell types) system for isolating early endosperm nuclei of Arabidopsis at high purity and how to generate parental-specific epigenome profiles. As a Protocol Extension, this article describes an adaptation of an existing Nature Protocol that details the use of the INTACT method for purification of root nuclei. We address how to obtain the INTACT lines, generate the starting material and purify the nuclei. We describe a method that allows purity assessment, which has not been previously addressed. The purified nuclei can be used for ChIP and DNA bisulfite treatment followed by next-generation sequencing (seq) to study histone modifications and DNA methylation profiles, respectively. By using two different Arabidopsis accessions as parents that differ by a large number of single-nucleotide polymorphisms (SNPs), we were able to distinguish the parental origin of epigenetic modifications. Our protocol describes the only working method to our knowledge for generating parental-specific epigenome profiles of the early Arabidopsis endosperm. The complete protocol, from silique collection to finished libraries, can be completed in 2 d for bisulfite-seq (BS-seq) and 3 to 4 d for ChIP-seq experiments.

This protocol is an extension to: Nat. Protoc. 6, 56–68 (2011); doi:10.1038/nprot.2010.175; published online 16 December 2010

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Figure 1: Experimental workflow and time estimates.
Figure 2: Generation of endosperm-specific INTACT lines with appropriate levels of NTF and BirA expression.
Figure 3: Homogenization method.
Figure 4: Different cross-combinations tested using INT lines.
Figure 5: Endosperm-specific ChIP results.

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Acknowledgements

This research was supported by a European Research Council Starting Independent Researcher grant (to C.K.), a grant from the Swedish Science Foundation (to C.K.), a grant from the Knut and Alice Wallenberg Foundation (to C.K.) and a grant from the Royal Physiographic Society in Lund (to J.M.-R.). We thank A. Ortiz Herrera for recording and editing the video.

Author information

Authors and Affiliations

  1. Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden

    Jordi Moreno-Romero, Juan Santos-González, Lars Hennig & Claudia Köhler

Authors
  1. Jordi Moreno-Romero
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  2. Juan Santos-González
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  3. Lars Hennig
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  4. Claudia Köhler
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Contributions

J.M.-R. and C.K. designed the research; J.M.-R. performed the experiments; J.M.-R., J.S.-G. and C.K. analyzed the data; L.H. developed the formula; and J.M.-R. and C.K. wrote the manuscript.

Corresponding authors

Correspondence to Jordi Moreno-Romero or Claudia Köhler.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Binding assay, yield and purity of INTACT purified crosslinked siliques

Comparison of INTACT purified nuclei from cross-linked and non-crosslinked siliques.

a. Bead-binding performed in nuclei extracted from cross-linked siliques (first two panels). Siliques that were either split open or left intact were cross-linked and efficient bead-binding was observed of nuclei extracted from this tissue. Nuclei from non-crosslinked siliques (last two panels) were binding to beads with similar efficiency.

b. (first and second columns) Amount of DNA recovered after purification (pg of DNA and nuclei number considering that an Arabidopsis 3n endosperm nuclei corresponds to 0.45 pg of DNA) and (last column) percentage of Ler left from a 43% (wt/wt) spike-in starting material. The starting material was 200 mg of INT siliques and 150 mg of Ler siliques.

Supplementary Figure 2 Ploidy analysis of the non-purified control sample (Step 17).

a. Ploidy profile of nuclei from adult leaves extracted by chopping ( Box 2 ). 2n, 4n and 8n peaks can be distinguished.

b. Ploidy profile of nuclei extracted from siliques at 4 DAP as described in the main procedure (Steps 1-17). Nuclei from maternal diploid tissue (2n, 4n and 8n) and endosperm nuclei (3n and 6n) can be detected as peaks. Quantifying this profile estimates about 16% of endosperm nuclei.

Supplementary information

Supplementary Figures and Text

Supplementary Figures 1 and 2, Supplementary Tables 1–3, and Supplementary Notes 1–3. (PDF 2908 kb)

Supplementary Data 1

bedGraph file used in Figure 5. (ZIP 24523 kb)

Supplementary Data 2

bedGraph file used in Figure 5. (ZIP 23040 kb)

Supplementary Data 3

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Supplementary Data 4

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Supplementary Data 5

bedGraph file used in Figure 5. (ZIP 23189 kb)

Supplementary Data 6

bedGraph file used in Figure 5. (ZIP 21828 kb)

Supplementary Data 7

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Supplementary Data 8

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Supplementary Data 9

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Supplementary Data 10

bedGraph file used in Figure 5. (ZIP 8028 kb)

Supplementary Data 11

bedGraph file used in Figure 5. (ZIP 8114 kb)

Supplementary Data 12

Archive containing custom scripts used in Figure 5 (script names and short descriptions listed in Supplementary Table 3). (ZIP 38056 kb)

Video showing nuclei purification (Steps 3–26) and sheared chromatin obtainment (Step 26B(i–viii). (MP4 29120 kb)

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Moreno-Romero, J., Santos-González, J., Hennig, L. et al. Applying the INTACT method to purify endosperm nuclei and to generate parental-specific epigenome profiles. Nat Protoc 12, 238–254 (2017). https://doi.org/10.1038/nprot.2016.167

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