Book/Journal Article

DKFZ-2026-02550

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Leveraging Single-Cell RNA Sequencing to Characterize Murine Ewing Sarcoma Xenografts.

Faehling, T. (First author)DKFZ* ; Yershova, A.DKFZ* ; Li, J. (Last author)DKFZ*

2027
Humana Press Totowa, NJ
ISBN: 978-1-0716-5586-3 (print), 978-1-0716-5587-0 (electronic)

Abstract: High-quality single-cell analyses critically depend on standardized tissue processing workflows that preserve cellular integrity and minimize dissociation-induced artifacts. Here, we describe a robust and reproducible protocol for the isolation, cryopreservation, and downstream processing of single-cell suspensions from Ewing sarcoma (EwS) tumors, including the bioinformatic analyses and tools to interrogate the resulting data. The workflow is optimized for strict cold-chain handling and employs a cold-active Bacillus licheniformis protease-based digestion strategy coupled with gentle mechanical dissociation to reduce cellular stress and transcriptional perturbations. Tumor tissue is processed under controlled low-temperature conditions, followed by filtration, cryopreservation, and storage compatible with single-cell RNA sequencing and multi-omics applications. This protocol supports high cell viability, preserves physiologically relevant transcriptional states, and provides a reproducible framework for downstream computational single-cell analyses of EwS tumor samples. The computational pipeline performs ambient RNA correction, barcode calling, doublet detection, SCTransform normalization, PCA/UMAP and clustering, and cell type annotation via the AUCell module scoring combined with multi-reference Azimuth label transfer. Cluster labels are validated by differential expression-based marker enrichment, and integrated embeddings are used for visualization. This end-to-end workflow ensures high cell viability, preserves physiologically relevant transcriptional states, and provides a reproducible framework for downstream computational single-cell analyses of EwS tumor samples.

Keyword(s): Animals (MeSH) ; Sarcoma, Ewing: genetics (MeSH) ; Sarcoma, Ewing: pathology (MeSH) ; Mice (MeSH) ; Single-Cell Analysis: methods (MeSH) ; Single-Cell Gene Expression Analysis (MeSH) ; Sequence Analysis, RNA: methods (MeSH) ; Humans (MeSH) ; Cryopreservation: methods (MeSH) ; Computational Biology: methods (MeSH) ; Cell Line, Tumor (MeSH) ; Ewing sarcoma ; Single-cell RNA sequencing ; Transcriptomics

Note: #LA:B410#LA:B410#

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Contributing Institute(s):

1. Translationale Pädiatrische Sarkomforschung (B410)
2. DKTK HD zentral (HD01)

Research Program(s):

1. 312 - Funktionelle und strukturelle Genomforschung (POF4-312) (POF4-312)

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Database coverage:
; SCOPUS

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