GSE200529
Dissociation Protocols used for Sarcoma Tissues Bias the Transcriptome observed in Single-cell and Single-nucleus RNA sequencing.
To determine whether sarcomas – tumors of mesenchymal origin – are subject to the same technical artifacts, we profiled patient-derived tumor explants (PDXs) propagated from three aggressive subtypes: osteosarcoma, Ewing sarcoma (ES), desmoplastic small round cell tumor (DSRCT). Given the rarity of these sarcoma subtypes, we explored whether single-nuclei RNA-seq from more widely available archival frozen specimens could accurately be identified by gene expression signatures linked to tissue phenotype or pathognomonic fusion proteins. Overall design: In this work, we studied sarcomas from varying tissue origins, including osteosarcoma (OS), Ewing's sarcoma (ES), and desmoplastic small round cell tumor (DSRCT). We used different dissociation protocols: Miltenyi Tumor Dissociation Kit, cold-active protease derived from Bacillus licheniformis, and Nuclei EZ Prep. These three protocols are described herein as Warm, Cold, and Nuclei protocols. For the OS specimens, we used the same Nuclei protocol and a different Warm protocol optimized for OS. For DSRCT and ES specimens, we performed the additional Cold protocol, using the cold-active protease, as we had more specimens available. Each sarcoma subtype included three PDX specimens derived from different patients. In total, we analyzed 125,831 whole-cells and nuclei across the three sarcoma subtypes and three dissociation protocols.