An optimized method to isolate intact nuclei from long-term frozen tissues for single-nucleus RNA sequencing

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, researchers at Hopp Children’s Cancer Center Heidelberg optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. They performed a technical comparison between different single nucleus RNA-sequencing (snRNA-seq) systems using a patient-derived xenograft model as a test sample. Further, they 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 this 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.

Isolation of intact nuclei from long-term frozen pediatric glioma tissue

(a) Schematic figure showing sample preparation steps. (b) Representative images showing the effect of an increasing number of washing steps on nuclei yield/integrity and amount of debris (scale bar 10 µm). (c) Nuclei yield decreases with increasing number of washes. (d) Staining for nuclear membrane, DNA and RNA reveals intact nuclei, with no leakage of nucleic acids (scale bars 5 µm). Nuclei originate from a pediatric pilocytic astrocytoma tissue frozen for seven years prior to nuclear extraction.