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EpiTyping: analysis of epigenetic aberrations in parental imprinting and X-chromosome inactivation using RNA-seq

Nature Protocols volume 18, pages 3881–3917 (2023)Cite this article

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Abstract

Human pluripotent stem cells (hPSCs) hold a central role in studying human development, in disease modeling and in regenerative medicine. These cells not only acquire genetic modifications when kept in culture, but they may also harbor epigenetic aberrations, mainly involving parental imprinting and X-chromosome inactivation. Here we present a detailed bioinformatic protocol for detecting such aberrations using RNA sequencing data. We provide a pipeline designed to process and analyze RNA sequencing data for the identification of abnormal biallelic expression of imprinted genes, and thus detect loss of imprinting. Furthermore, we show how to differentiate among X-chromosome inactivation, full activation and aberrant erosion of X chromosome in female hPSCs. In addition to providing bioinformatic tools, we discuss the impact of such epigenetic variations in hPSCs on their utility for various purposes. This pipeline can be used by any user with basic understanding of the Linux command line. It is available on GitHub as a software container (https://github.com/Gal-Keshet/EpiTyping) and produces reliable results in 1–4 d.

Key points

  • This protocol provides a step-by-step procedure to infer the presence of epigenetic aberrations in human pluripotent stem cells from RNA sequencing data, specifically focusing on the identification of imprinting and X-inactivation status.

  • The pipeline provides a simple and easy-to-use methodology for the reliable identification of gene- and locus-specific loss-of-imprinting events as well as the accurate discrimination among different X-inactivation statuses.

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Fig. 1: Epigenetic aberrations.
Fig. 2: Expression and allelic representation of epigenetic aberrations.
Fig. 3: Workflow.
Fig. 4: Example of output.
Fig. 5: Implications of epigenetic aberrations.

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Data availability

The RNA-seq samples used as an example in this protocol, along with additional samples, can be retrieved from the SRA database. Their accession numbers are specified in Supplementary Table 2.

Code availability

All code used in this protocol is available at https://github.com/Gal-Keshet/EpiTyping and in the Supplementary Information.

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Acknowledgements

We thank Assa Sherman for additional testing of the pipeline and providing constructive input, and all members of The Azrieli Center for Stem Cells and Genetic Research for critical reading of the manuscript. This work was partially supported by the Azrieli Foundation, the Rosetrees Trust, the US–Israel Binational Science Foundation (2021278), the Israel Science Foundation (2054/22), the ISF-Israel Precision Medicine Partnership (IPMP) Program (3605/21) and the HEAL project, funded by the European Union, EU Horizon (101056712). Views and opinions expressed are, however, those of the authors only and do not necessarily reflect those of the European Union or EU Horizon. Neither the European Union nor EU Horizon can be held responsible for them. N.B. is the Herbert Cohn Chair in Cancer Research.

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Author notes

  1. These authors contributed equally: Roni Sarel-Gallily, Gal Keshet.

Authors and Affiliations

  1. The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

    Roni Sarel-Gallily, Gal Keshet, Shay Kinreich, Guy Haim-Abadi & Nissim Benvenisty

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  1. Roni Sarel-Gallily
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Contributions

R.S.-G., G.K. and N.B. designed the analysis. R.S.-G. developed the X-inactivation analysis bioinformatic pipeline. G.K. developed the LOI analysis bioinformatic pipeline. R.S.-G. and G.K. wrote the manuscript and designed the automated code with input from S.K, G.H.-A. and N.B. N.B. supervised the work.

Corresponding authors

Correspondence to Gal Keshet or Nissim Benvenisty.

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Competing interests

N.B. is chief scientific officer of NewStem Ltd.

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Nature Protocols thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Bar, S. & Benvenisty, N. EMBO J. 38, e101033 (2019): https://doi.org/10.15252/embj.2018101033

Keshet, G. & Benvenisty, N. Stem Cell Rep. 16, 2520–2533 (2021): https://doi.org/10.1016/j.stemcr.2021.09.002

Sarel-Gallily, R. & Benvenisty, N. Cells 11, 1729 (2022): https://doi.org/10.3390/cells11111729

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Files 1–5.

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Sarel-Gallily, R., Keshet, G., Kinreich, S. et al. EpiTyping: analysis of epigenetic aberrations in parental imprinting and X-chromosome inactivation using RNA-seq. Nat Protoc 18, 3881–3917 (2023). https://doi.org/10.1038/s41596-023-00898-5

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