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Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues

Kati J. Ernst, Konstantin Okonechnikov, Josephine Bageritz, Jan-Philipp Mallm, Andrea Wittmann, Kendra K. Maaß, Svenja Leible, View ORCID ProfileMichael Boutros, Stefan M. Pfister, Marc Zuckermann, David T.W. Jones
doi: https://doi.org/10.1101/2020.10.23.351809
Kati J. Ernst
1Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany
2Pediatric Glioma Research Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Konstantin Okonechnikov
1Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany
3Division of Pediatric Neuro-oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Josephine Bageritz
4Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Jan-Philipp Mallm
5Single-Cell Open Lab, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Andrea Wittmann
1Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany
2Pediatric Glioma Research Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Kendra K. Maaß
1Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany
3Division of Pediatric Neuro-oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Svenja Leible
4Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Michael Boutros
4Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • ORCID record for Michael Boutros
Stefan M. Pfister
1Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany
3Division of Pediatric Neuro-oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
6Department of Pediatric Hematology and Oncology, University Hospital, Heidelberg, Germany
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Marc Zuckermann
1Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany
3Division of Pediatric Neuro-oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • For correspondence: david.jones@kitz-heidelberg.de m.zuckermann@kitz-heidelberg.de
David T.W. Jones
1Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany
2Pediatric Glioma Research Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • For correspondence: david.jones@kitz-heidelberg.de m.zuckermann@kitz-heidelberg.de
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Abstract

Recent advances allowing the genomic analysis of individual cells from a bulk population have provided intriguing new insights into areas such as developmental processes and tumor heterogeneity. Most approaches to date, however, rely on the availability of fresh surgical specimens, thereby dramatically reducing the ability to profile particularly rare tissue types. Pediatric central nervous system tumors – the leading cause of childhood cancer deaths – represent one such example, where often only frozen rather than native material is available. Due to an increasing need for advanced techniques to understand the heterogeneity of these tumors, we optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. We performed a technical comparison between different single nucleus RNA-sequencing (snRNA-seq) systems using a patient-derived xenograft model as a test sample. Further, we applied the established nucleus isolation method to analyze frozen primary tissue from two pediatric central nervous system tumors – one pilocytic astrocytoma and one glioblastoma – allowing the identification of distinct tumor cell populations and infiltrating microglia. The results show that our fast, simple and low-cost nuclear isolation protocol provides intact nuclei, which can be used in both droplet-based 3’ transcriptome amplification (10X Genomics) and plate-based whole transcriptome amplification (Fluidigm C1) single-cell sequencing platforms, thereby dramatically increasing the potential for application of such methods to rare entities.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted October 23, 2020.
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Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues
Kati J. Ernst, Konstantin Okonechnikov, Josephine Bageritz, Jan-Philipp Mallm, Andrea Wittmann, Kendra K. Maaß, Svenja Leible, Michael Boutros, Stefan M. Pfister, Marc Zuckermann, David T.W. Jones
bioRxiv 2020.10.23.351809; doi: https://doi.org/10.1101/2020.10.23.351809
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Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues
Kati J. Ernst, Konstantin Okonechnikov, Josephine Bageritz, Jan-Philipp Mallm, Andrea Wittmann, Kendra K. Maaß, Svenja Leible, Michael Boutros, Stefan M. Pfister, Marc Zuckermann, David T.W. Jones
bioRxiv 2020.10.23.351809; doi: https://doi.org/10.1101/2020.10.23.351809

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