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A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors

Michal Slyper, Caroline B. M. Porter, Orr Ashenberg, Julia Waldman, Eugene Drokhlyansky, Isaac Wakiro, Christopher Smillie, Gabriela Smith-Rosario, Jingyi Wu, Danielle Dionne, Sébastien Vigneau, Judit Jané-Valbuena, Sara Napolitano, Mei-Ju Su, Anand G. Patel, Asa Karlstrom, Simon Gritsch, Masashi Nomura, Avinash Waghray, Satyen H. Gohil, Alexander M. Tsankov, Livnat Jerby-Arnon, Ofir Cohen, Johanna Klughammer, Yanay Rosen, Joshua Gould, Bo Li, Lan Nguyen, Catherine J. Wu, Benjamin Izar, Rizwan Haq, F. Stephen Hodi, Charles H. Yoon, Aaron N. Hata, Suzanne J. Baker, Mario L. Suvà, Raphael Bueno, Elizabeth H. Stover, Ursula A. Matulonis, Michael R. Clay, Michael A. Dyer, Natalie B. Collins, Nikhil Wagle, Asaf Rotem, Bruce E. Johnson, Orit Rozenblatt-Rosen, Aviv Regev
doi: https://doi.org/10.1101/761429
Michal Slyper
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Caroline B. M. Porter
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Orr Ashenberg
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Julia Waldman
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Eugene Drokhlyansky
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Isaac Wakiro
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Christopher Smillie
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Gabriela Smith-Rosario
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Jingyi Wu
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Danielle Dionne
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Sébastien Vigneau
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Judit Jané-Valbuena
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Sara Napolitano
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Mei-Ju Su
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Anand G. Patel
3Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
4Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
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Asa Karlstrom
3Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
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Simon Gritsch
5Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
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Masashi Nomura
5Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
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Avinash Waghray
6Massachusetts General Hospital, Harvard University, Boston, MA 02114, USA
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Satyen H. Gohil
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Alexander M. Tsankov
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Livnat Jerby-Arnon
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Ofir Cohen
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Johanna Klughammer
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Yanay Rosen
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Joshua Gould
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Bo Li
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
8Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA
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Lan Nguyen
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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Catherine J. Wu
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
9Department of Internal Medicine, Brigham and Women’s Hospital, Boston, MA 02215, USA; Harvard Medical School, Boston, MA 02215, USA
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Benjamin Izar
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
10Laboratory for Systems Pharmacology, Harvard Medical School, Boston, MA 02114, USA; Center for Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
11Ludwig Center for Cancer Research at Harvard, Boston, MA, USA
12Melanoma Disease Center, Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Rizwan Haq
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
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F. Stephen Hodi
7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
12Melanoma Disease Center, Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Charles H. Yoon
7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
13Brigham and Women’s Hospital, Department of Surgical Oncology, Boston, MA 02215, USA
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Aaron N. Hata
14Massachusetts General Cancer Center, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
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Suzanne J. Baker
3Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
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Mario L. Suvà
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
5Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
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Raphael Bueno
15Division of Thoracic Surgery, Brigham and Women’s Hospital, Boston, MA 02215, USA
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Elizabeth H. Stover
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Ursula A. Matulonis
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Michael R. Clay
16Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA
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Michael A. Dyer
3Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
17Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
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Natalie B. Collins
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
18Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Division of Pediatric Hematology and Oncology, Children’s Hospital, Boston, MA 02215, USA
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Nikhil Wagle
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Asaf Rotem
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Bruce E. Johnson
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA 02215, USA
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Orit Rozenblatt-Rosen
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
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  • For correspondence: orit@broadinstitute.org aregev@broadinstitute.org
Aviv Regev
1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
17Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
19Koch Institute of Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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  • For correspondence: orit@broadinstitute.org aregev@broadinstitute.org
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Abstract

Single cell genomics is essential to chart the complex tumor ecosystem. While single cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumor tissues, single nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each strategy requires modifications to fit the unique characteristics of different tissue and tumor types, posing a barrier to adoption. Here, we developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We tested eight tumor types of varying tissue and sample characteristics (resection, biopsy, ascites, and orthotopic patient-derived xenograft): lung cancer, metastatic breast cancer, ovarian cancer, melanoma, neuroblastoma, pediatric sarcoma, glioblastoma, pediatric high-grade glioma, and chronic lymphocytic leukemia. Analyzing 212,498 cells and nuclei from 39 clinical samples, we evaluated protocols by cell quality, recovery rate, and cellular composition. We optimized protocols for fresh tissue dissociation for different tumor types using a decision tree to account for the technical and biological variation between clinical samples. We established methods for nucleus isolation from OCT embedded and fresh-frozen tissues, with an optimization matrix varying mechanical force, buffer, and detergent. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types and intrinsic expression profiles, but at different proportions. Our work provides direct guidance across a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

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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 4.0 International license.
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A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors
Michal Slyper, Caroline B. M. Porter, Orr Ashenberg, Julia Waldman, Eugene Drokhlyansky, Isaac Wakiro, Christopher Smillie, Gabriela Smith-Rosario, Jingyi Wu, Danielle Dionne, Sébastien Vigneau, Judit Jané-Valbuena, Sara Napolitano, Mei-Ju Su, Anand G. Patel, Asa Karlstrom, Simon Gritsch, Masashi Nomura, Avinash Waghray, Satyen H. Gohil, Alexander M. Tsankov, Livnat Jerby-Arnon, Ofir Cohen, Johanna Klughammer, Yanay Rosen, Joshua Gould, Bo Li, Lan Nguyen, Catherine J. Wu, Benjamin Izar, Rizwan Haq, F. Stephen Hodi, Charles H. Yoon, Aaron N. Hata, Suzanne J. Baker, Mario L. Suvà, Raphael Bueno, Elizabeth H. Stover, Ursula A. Matulonis, Michael R. Clay, Michael A. Dyer, Natalie B. Collins, Nikhil Wagle, Asaf Rotem, Bruce E. Johnson, Orit Rozenblatt-Rosen, Aviv Regev
bioRxiv 761429; doi: https://doi.org/10.1101/761429
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A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors
Michal Slyper, Caroline B. M. Porter, Orr Ashenberg, Julia Waldman, Eugene Drokhlyansky, Isaac Wakiro, Christopher Smillie, Gabriela Smith-Rosario, Jingyi Wu, Danielle Dionne, Sébastien Vigneau, Judit Jané-Valbuena, Sara Napolitano, Mei-Ju Su, Anand G. Patel, Asa Karlstrom, Simon Gritsch, Masashi Nomura, Avinash Waghray, Satyen H. Gohil, Alexander M. Tsankov, Livnat Jerby-Arnon, Ofir Cohen, Johanna Klughammer, Yanay Rosen, Joshua Gould, Bo Li, Lan Nguyen, Catherine J. Wu, Benjamin Izar, Rizwan Haq, F. Stephen Hodi, Charles H. Yoon, Aaron N. Hata, Suzanne J. Baker, Mario L. Suvà, Raphael Bueno, Elizabeth H. Stover, Ursula A. Matulonis, Michael R. Clay, Michael A. Dyer, Natalie B. Collins, Nikhil Wagle, Asaf Rotem, Bruce E. Johnson, Orit Rozenblatt-Rosen, Aviv Regev
bioRxiv 761429; doi: https://doi.org/10.1101/761429

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