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Nanogrid single-nucleus RNA sequencing reveals phenotypic diversity in breast cancer.

ABSTRACT: Single cell RNA sequencing has emerged as a powerful tool for resolving transcriptional diversity in tumors, but is limited by throughput, cost and the ability to process archival frozen tissue samples. Here we develop a high-throughput 3' single-nucleus RNA sequencing approach that combines nanogrid technology, automated imaging, and cell selection to sequence up to ~1800 single nuclei in parallel. We compare the transcriptomes of 485 single nuclei to 424 single cells in a breast cancer cell line, which shows a high concordance (93.34%) in gene levels and abundance. We also analyze 416 nuclei from a frozen breast tumor sample and 380 nuclei from normal breast tissue. These data reveal heterogeneity in cancer cell phenotypes, including angiogenesis, proliferation, and stemness, and a minor subpopulation (19%) with many overexpressed cancer genes. Our studies demonstrate the utility of nanogrid single-nucleus RNA sequencing for studying the transcriptional programs of tumor nuclei in frozen archival tissue samples.Single cell RNA sequencing is a powerful tool for understanding cellular diversity but is limited by cost, throughput and sample preparation. Here the authors use nanogrid technology with integrated imaging to sequence thousands of cancer nuclei in parallel from fresh or frozen tissue.

SUBMITTER: Gao R

PROVIDER: S-EPMC5550415 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Nanogrid single-nucleus RNA sequencing reveals phenotypic diversity in breast cancer.

Gao Ruli R Kim Charissa C Sei Emi E Foukakis Theodoros T Crosetto Nicola N Chan Leong-Keat LK Srinivasan Maithreyan M Zhang Hong H Meric-Bernstam Funda F Navin Nicholas N

Nature communications 20170809 1

Single cell RNA sequencing has emerged as a powerful tool for resolving transcriptional diversity in tumors, but is limited by throughput, cost and the ability to process archival frozen tissue samples. Here we develop a high-throughput 3' single-nucleus RNA sequencing approach that combines nanogrid technology, automated imaging, and cell selection to sequence up to ~1800 single nuclei in parallel. We compare the transcriptomes of 485 single nuclei to 424 single cells in a breast cancer cell li ...[more]

PMID: 28794488

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Project description:BackgroundSingle cell sequencing of human heart tissue is technically challenging and methods to cryopreserve heart tissue for obtaining single cell information have not been standardized. Studies published to date have used varying methods to preserve and process human heart tissue, and have generated interesting datasets, but development of a biobanking standard has not yet been achieved. Heart transcription patterns are known to be regionally diverse, and there are few single cell datasets for normal human heart tissue.MethodsUsing pig tissue, we developed a rigorous and reproducible method for tissue mincing and cryopreservation that allowed recovery of high quality single nuclei RNA. We subsequently tested this protocol on normal human heart tissue obtained from organ donors and were able to recover high quality nuclei for generation of single nuclei RNA-seq datasets, using a commercially available platform from 10× Genomics. We analyzed these datasets using standard software packages such as CellRanger and Seurat.ResultsHuman heart tissue preserved with our method consistently yielded nuclear RNA with RNA Integrity Numbers of greater than 8.5. We demonstrate the utility of this method for single nuclei RNA-sequencing of the normal human interventricular septum and delineating its cellular diversity. The human IVS showed unexpected diversity with detection of 23 distinct cell clusters that were subsequently categorized into different cell types. Cardiomyocytes and fibroblasts were the most commonly identified cell types and could be further subdivided into 5 different cardiomyocyte subtypes and 6 different fibroblast subtypes that differed by gene expression patterns. Ingenuity Pathway analysis of these gene expression patterns suggested functional diversity in these cell subtypes.ConclusionsHere we report a simple technical method for cryopreservation and subsequent nuclear isolation of human interventricular septum tissue that can be done with common laboratory equipment. We show how this method can be used to generate single nuclei transcriptomic datasets that rival those already published by larger groups in terms of cell diversity and complexity and suggest that this simple method can provide guidance for biobanking of human myocardial tissue for complex genomic analysis.

Dataset Information

Nanogrid single-nucleus RNA sequencing reveals phenotypic diversity in breast cancer.

Publications

Nanogrid single-nucleus RNA sequencing reveals phenotypic diversity in breast cancer.

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