Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing
Year: 2025
Type: article
Abstract: 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.
Source: BIO-PROTOCOL
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W4410844175