Abstract
Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in 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.
Highlights
Tumors encompass complex cellular ecosystems of malignant and non-malignant cells, whose diversity and interactions affect cancer progression and drug response and resistance
We provide a comprehensive analysis summary for each sample tested in a dedicated website
For ‘cellular composition’, we considered the diversity of cell types captured, the proportion of cells/nuclei recovered from each subset and the copy number aberration (CNA) pattern classes that are recovered in malignant cells
Summary
Tumors encompass complex cellular ecosystems of malignant and non-malignant cells, whose diversity and interactions affect cancer progression and drug response and resistance. Nuclei have lower amounts of mRNA compared to cells and are more challenging to enrich or deplete for specific cell types of interest. Both scRNA-Seq and snRNA-Seq pose experimental challenges when applied to different tumor types, due to the distinct cellular composition and extracellular matrix (ECM) in different tumors, and each assay requires dedicated customizations[11]. To address these challenges, we developed a systematic toolbox for fresh and frozen tumor processing using scRNA-Seq and snRNA-Seq, respectively (Fig. 1a). Our work provides direct recommended protocols for multiple tumor types, decision trees that allow researchers to choose the most suitable protocol for their research goals, and guidelines on how to customize protocols for new tumor and specimen types
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