Optimized protocol for single-cell and bulk transcriptomic profiling of stabilized hemocytes from Anopheles gambiae mosquitoes

Abstract Background Insect hemocytes play a crucial role in the innate immune response against pathogens. However, transcriptomic profiling and analysis of hemocytes in insect vectors, such as Anopheles gambiae mosquitoes, are challenging due to their small size, low RNA content, limited numbers, and highly reactive nature. Methods To address these challenges, we developed a protocol for single-cell and bulk RNA sequencing of stabilized hemocytes from adult female An. gambiae mosquitoes. Immediate stabilization of hemocytes following perfusion prevents transcriptional changes during sample collection and RNA degradation, thereby minimizing technical artifacts. After stabilization, hemocytes are filtered to remove contaminating tissue and ambient RNA introduced by tissue injury during sample collection, enriching for hemocyte-specific transcripts. The stabilized hemocytes can be stored for up to four weeks, allowing for convenient sample storage, transportation, and simultaneous processing of multiple samples, which reduces costs and minimizes batch effects. Discussion Our single-cell RNA sequencing stabilization and processing protocol is compatible with the 10X Genomics platform, yielding high-quality and reproducible data. We demonstrate higher hemocyte recovery compared to other contemporary methods, enabling the identification of rare hemocyte populations. However, this protocol requires further optimization to increase the recovery of small-sized prohemocytes (< 5 μm in diameter). Our bulk mRNA stabilization and hemocyte enrichment protocol yields high-quality, intact RNA, and Illumina bulk mRNA sequencing libraries enable an in-depth investigation of gene expression changes in hemocytes. These protocols significantly improve transcriptomic analysis of hemocytes, which is critical for advancing discoveries in vector biology and disease control. Moreover, the protocol can be applied to other mosquito species and likely many other insects with only minor optimizations.