Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing

Single-cell RNA sequencing has revolutionized molecular cell biology by enabling the identification of unique transcription profiles and cell transcription states within the same tissue. However, tissue dissociation presents a challenge for non-model organisms, as commercial kits are often incompatible, and current protocols rely on tissue enzymatic digestion for extended periods. Tissue digestion can alter cell transcription in response to temperature and the stress caused by enzymatic treatment. Here, we propose a protocol to stabilize RNA using a deep eutectic solvent (Vivophix, Rapid Labs) prior to tissue dissociation, thereby avoiding transcription changes induced by the process and preventing RNase activity during incubation. We validated this methodology for three medically important insect vectors: <i>Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis.</i> Single-cell RNA sequencing using our insect midgut dissociation protocol yielded high-quality sequencing results, with a high number of cells recovered, a low percentage of mitochondrial reads, and a low percentage of ambient RNA-two hallmark standards of cell quality. Key features • This protocol stabilizes tissue RNA before dissociation, avoiding RNase-mediated RNA degradation and transcription changes during the dissociation process. • Current protocols for insect midgut dissociation use enzymatic digestion of live tissues at temperatures that are incompatible with insect physiology. • We validated this new stabilization-dissociation methodology for insect midgut, yielding high-quality single-cell RNA sequencing with high gene counts, low mitochondrial RNA, and minimal ambient RNA contamination. Graphical overview Mosquito and sand fly midgut RNA stabilization and dissociation protocol for single-cell sequencing applications.