Background

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular heterogeneity by enabling high-resolution molecular profiling at the level of individual cells. While conventional bulk RNA sequencing offers deep insights into transcription regulation, it only captures the average gene expression across a population of cells, masking potential cell-to-cell variability. In contrast, single-cell omics allow for the analysis of transcriptomes from individual cells, generating distinct gene expression profiles that reveal cell-specific functions and dynamic states within complex tissues.

Mosquitoes are the main vectors of arboviruses and parasites, impacting millions of people each year. Blood feeding is a critical behavior in female mosquitoes, as it provides the nutrients necessary for egg development; however, it also enables the transmission of pathogens when mosquitoes feed on infected hosts. The ingestion of a blood meal triggers profound physiological changes, particularly in the fat body, a central metabolic and reproductive tissue in mosquitoes [1–3]. Functionally similar to the liver and adipose tissue in mammals, the fat body rapidly shifts its gene expression after a blood meal, including the production of yolk proteins like vitellogenin that are essential for reproduction. Trophocytes—large cells with the cytoplasm filled with extensive lipid droplets—constitute the main cell type of the fat body [1–4]. These are adipocyte-like cells with high lipid content and a fragile structure, which makes dissociation and single-cell isolation very difficult, leading to poor cell recovery, low viability, and reduced RNA integrity when using standard single-cell isolation procedures.

A critical step in scRNA-seq is an efficient dissociation of tissues into a high-quality single-cell suspension. However, certain tissue types—such as adipose tissue—pose a significant technical challenge due to their large size (20–300 μm) [5], structural fragility, and high lipid content [6]. Enzymatic dissociation methods (e.g., collagenase or trypsin digestion) are often used to dissociate tissues, but they can introduce technical artifacts, damage fragile cells, and reduce RNA integrity, especially in lipid-rich tissues [6–8]. These limitations may result in poor cell viability and biased transcriptomic profiles, making conventional scRNA-seq challenging for these tissues. Single-nucleus RNA sequencing (snRNA-seq) offers several advantages for challenging tissues, including those that are fragile, lipid-rich, or difficult to dissociate. snRNA-seq avoids dissociation-induced transcriptional artifacts, preserves representation of large or fragile cell types, and is compatible with frozen or archived tissues [9,10]. However, because nuclear RNA is enriched for pre-mRNAs and lacks some cytoplasmic transcripts, snRNA-seq typically detects fewer genes per nucleus and may underestimate transcripts with strong cytoplasmic localization [11,12]. Thus, while snRNA-seq provides a powerful alternative when whole-cell isolation is not feasible, it also requires accounting for nuclear-specific transcriptional features during data analysis.

Prior studies have explored single-nucleus transcriptomic approaches in insects. In Aedes aegypti, single-nucleus RNA sequencing has been applied to both brain and midgut tissues, demonstrating the feasibility of nuclear profiling in mosquitoes [13,14]. However, quality metrics such as nuclei yield, ambient RNA contamination, and percentage of mitochondrial transcripts are poorly reported. Additionally, snRNA-seq has been performed in the fruit fly Drosophila melanogaster and the Bombyx mori moth fat body tissues, profiling cell-type diversity and functional heterogeneity [15,16]. An optimized Drosophila nuclei isolation protocol reported the recovery of ~10⁶ nuclei from 40 fat body tissues, with improved levels of mitochondrial contamination (<20%) and unique transcripts (>200) compared to enzymatic methods [17]. Despite these advances, isolating intact cells from the mosquito fat body remains technically challenging. To date, no standardized or optimized nuclei isolation protocol has been developed for Anopheles gambiae fat body. Here, we present a reproducible nuclei-based method that yields high-quality, intact nuclei from Anopheles gambiae fat body with consistent recovery across samples. Our protocol recovers approximately 1 × 10⁶ nuclei from 25 body wall tissues, maintains nuclear membrane integrity, and supports low levels of ambient RNA contamination, enabling robust snRNA-seq profiling. In addition, the workflow is compatible with both fresh and methanol-stabilized tissues and is completed rapidly without enzymatic digestion. These features collectively provide a practical and reliable approach for high-quality transcriptomic analysis of this metabolically and immunologically important tissue.