RT Journal Article
SR Electronic
T1 A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 761429
DO 10.1101/761429
A1 Slyper, Michal
A1 Porter, Caroline B. M.
A1 Ashenberg, Orr
A1 Waldman, Julia
A1 Drokhlyansky, Eugene
A1 Wakiro, Isaac
A1 Smillie, Christopher
A1 Smith-Rosario, Gabriela
A1 Wu, Jingyi
A1 Dionne, Danielle
A1 Vigneau, Sébastien
A1 Jané-Valbuena, Judit
A1 Napolitano, Sara
A1 Su, Mei-Ju
A1 Patel, Anand G.
A1 Karlstrom, Asa
A1 Gritsch, Simon
A1 Nomura, Masashi
A1 Waghray, Avinash
A1 Gohil, Satyen H.
A1 Tsankov, Alexander M.
A1 Jerby-Arnon, Livnat
A1 Cohen, Ofir
A1 Klughammer, Johanna
A1 Rosen, Yanay
A1 Gould, Joshua
A1 Li, Bo
A1 Nguyen, Lan
A1 Wu, Catherine J.
A1 Izar, Benjamin
A1 Haq, Rizwan
A1 Hodi, F. Stephen
A1 Yoon, Charles H.
A1 Hata, Aaron N.
A1 Baker, Suzanne J.
A1 Suvà, Mario L.
A1 Bueno, Raphael
A1 Stover, Elizabeth H.
A1 Matulonis, Ursula A.
A1 Clay, Michael R.
A1 Dyer, Michael A.
A1 Collins, Natalie B.
A1 Wagle, Nikhil
A1 Rotem, Asaf
A1 Johnson, Bruce E.
A1 Rozenblatt-Rosen, Orit
A1 Regev, Aviv
YR 2019
UL http://biorxiv.org/content/early/2019/09/12/761429.abstract
AB 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.