A simplified preparation method for single-nucleus RNA-sequencing using long-term frozen brain tumor tissues

Area of Science:

• Oncology
• Genomics
• Neuroscience

Background:

• Single-cell RNA-sequencing (scRNA-seq) offers valuable insights into developmental biology and tumor heterogeneity.
• Current scRNA-seq methods often require fresh surgical specimens, limiting the analysis of rare tissue types.

Purpose of the Study:

• To optimize a method for isolating intact nuclei from long-term frozen pediatric glioma tissues.
• To enable single-nucleus RNA-sequencing (snRNA-seq) on frozen brain tumor samples, expanding research possibilities for rare tumor entities.

Main Methods:

• Optimized a protocol for isolating intact nuclei from frozen pediatric glioma tissues.
• Conducted a technical comparison of different single-nucleus RNA-sequencing (snRNA-seq) platforms.
• Applied the nucleus isolation method to analyze frozen primary glioma tissues.
• Incorporated shorter RNA fragments into the 3' sequencing library for improved gene detection.

Main Results:

• The optimized protocol yields intact nuclei suitable for both droplet- and plate-based snRNA-seq.
• Analysis of frozen glioma tissues identified distinct tumor cell populations and infiltrating microglia.
• Improved gene detection and cell type annotation were achieved through library preparation optimization.

Conclusions:

• The developed nuclear isolation protocol is fast, simple, and cost-effective.
• This method significantly enhances the potential for studying rare tumor entities using frozen brain tumor tissue.
• The protocol is specifically tailored for analyzing frozen pediatric brain tumor samples with snRNA-seq.