Abstract Recent advances allowing the genomic analysis of individual cells from a bulk population have provided intriguing new insights into areas such as developmental processes and tumor heterogeneity. Most approaches to date, however, rely on the availability of fresh surgical specimens, thereby dramatically reducing the ability to profile particularly rare tissue types. Pediatric central nervous system tumors – the leading cause of childhood cancer deaths – represent one such example, where often only frozen rather than native material is available. Due to an increasing need for advanced techniques to understand the heterogeneity of these tumors, we optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. We performed a technical comparison between different single nucleus RNA-sequencing (snRNA-seq) systems using a patient-derived xenograft model as a test sample. Further, we applied the established nucleus isolation method to analyze frozen primary tissue from two pediatric central nervous system tumors – one pilocytic astrocytoma and one glioblastoma – allowing the identification of distinct tumor cell populations and infiltrating microglia. The results show that our fast, simple and low-cost nuclear isolation protocol provides intact nuclei, which can be used in both droplet-based 3’ transcriptome amplification (10X Genomics) and plate-based whole transcriptome amplification (Fluidigm C1) single-cell sequencing platforms, thereby dramatically increasing the potential for application of such methods to rare entities.

Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues