Abstract
Background Single-cell RNA-seq has emerged as an innovative technology used to study complex tissues and characterize cell types, states, and lineages at a single-cell level. Rapid adoption of this technology has led to a flurry of research creating single-cell atlases for many organs, cancers, and developmental models. Despite the tremendous success of this technology, sarcomas, rare cancers of mesenchymal origin, have not yet widely benefited from the adoption of single-cell RNA-seq. Due to their rarity, obtaining fresh clinical specimens is challenging. Single-nucleus RNA-seq could remove limitations from obtaining fresh tissue and enable immediate processing of archival tissue. However, biases may exist during dissociation of either fresh or frozen specimens that can hinder reproducible results and introduce technical or biological artifacts.
Results To address these questions, we systematically assessed dissociation methods across different sarcoma subtypes. Here, we compare gene expression from single-cell and single-nucleus RNA-sequencing of 125,831 whole-cells and nuclei from Ewing’s sarcoma, desmoplastic small round cell tumor, and osteosarcoma patient-derived xenografts. We detected warm dissociation artifacts from single-cell samples and gene length bias in single-nucleus samples. Classic sarcoma gene signatures were observed regardless of dissociation method. Finally, we show that dissociation method biases can be computationally corrected.
Conclusions We highlighted transcriptional biases, including warm dissociation and gene-length biases, introduced by any dissociation method for various sarcoma subtypes. This work will inform researchers on choice of dissociation method and careful interpretation of data due to dissociation biases.
Competing Interest Statement
The authors have declared no competing interest.
