Optimized Method for High-Quality Isolation of Single-Nuclei From Mosquito Fat Body for RNA Sequencing.

Area of Science:

• Molecular Biology
• Genomics
• Insect Physiology

Background:

• Single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) are powerful tools for understanding cellular heterogeneity.
• Tissue dissociation for scRNA-seq/snRNA-seq is challenging for non-model organisms and lipid-rich tissues.
• The mosquito fat body presents unique challenges due to fragile trophocytes and high lipid content.

Purpose of the Study:

• To develop and optimize a reproducible method for isolating high-quality nuclei from the Anopheles gambiae mosquito fat body.
• To enable high-throughput transcriptomic analysis of mosquito fat body cells using single-nucleus RNA sequencing (snRNA-seq).
• To address limitations of conventional dissociation methods for lipid-rich and fragile tissues.

Main Methods:

• Optimized nuclei isolation protocol involving methanol fixation and lipid removal.
• Cell lysis followed by nuclei purification using a sucrose cushion.
• Validation of the protocol on both sugar-fed and blood-fed Anopheles gambiae samples.

Main Results:

• The optimized protocol efficiently isolates nuclei from the fragile, lipid-rich mosquito fat body.
• Single-nucleus RNA sequencing (snRNA-seq) data generated using this method exhibit high quality, characterized by high gene counts and low mitochondrial RNA content.
• Established quality metrics effectively filter ambient RNA contamination, improving transcriptomic accuracy.

Conclusions:

• This optimized nuclei isolation methodology enables high-quality snRNA-seq from the Anopheles gambiae fat body.
• The protocol is robust across different physiological states (sugar-fed vs. blood-fed).
• This approach facilitates deeper understanding of mosquito fat body cellular functions and gene expression patterns.