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.
Highlights
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
We developed a nanogrid platform and microfluidic depositing system that enables imaging, selection, and sequencing of thousands of single cells or nuclei in parallel. We applied this nanogrid single-nucleus RNA sequencing (RNA-seq) (SNRS) system to compare the transcriptional profiles of cancer cells and nuclei in cell lines and further applied this method to study phenotypic diversity and subpopulations in a frozen tumor sample from a triple-negative breast cancer (TNBC) patient
Prior to single cell analysis, we investigated whether the transcriptional profiles of bulk cells and nuclear fractions are concordant in breast cancer cell lines
Summary
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. To date, no one has investigated the transcriptional profiles of single tumor nuclei, to determine if they are representative of whole tumor cells To address these limitations, we developed a nanogrid platform and microfluidic depositing system that enables imaging, selection, and sequencing of thousands of single cells or nuclei in parallel. We developed a nanogrid platform and microfluidic depositing system that enables imaging, selection, and sequencing of thousands of single cells or nuclei in parallel We applied this nanogrid single-nucleus RNA-seq (SNRS) system to compare the transcriptional profiles of cancer cells and nuclei in cell lines and further applied this method to study phenotypic diversity and subpopulations in a frozen tumor sample from a triple-negative breast cancer (TNBC) patient
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