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 incompati...
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| Format: | Article |
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Bio-protocol LLC
2025-06-01
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| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5352&type=0 |
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| author | Ana Barletta Octavio Talyuli Pedro Cecilio Carolina Barillas-Mury |
| author_facet | Ana Barletta Octavio Talyuli Pedro Cecilio Carolina Barillas-Mury |
| author_sort | Ana Barletta |
| collection | DOAJ |
| description | 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: Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis. 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. |
| format | Article |
| id | doaj-art-8018bda95b2d42d2943d9427196bc148 |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-8018bda95b2d42d2943d9427196bc1482025-08-20T02:20:52ZengBio-protocol LLCBio-Protocol2331-83252025-06-01151210.21769/BioProtoc.5352Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA SequencingAna Barletta0Octavio Talyuli1Pedro Cecilio2Carolina Barillas-Mury3Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health; Rockville, MD, USALaboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health; Rockville, MD, USALaboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health; Rockville, MD, USALaboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health; Rockville, MD, USASingle-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: Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis. 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.https://bio-protocol.org/en/bpdetail?id=5352&type=0 |
| spellingShingle | Ana Barletta Octavio Talyuli Pedro Cecilio Carolina Barillas-Mury Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing Bio-Protocol |
| title | Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing |
| title_full | Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing |
| title_fullStr | Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing |
| title_full_unstemmed | Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing |
| title_short | Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing |
| title_sort | optimized midgut tissue dissociation of mosquitoes and sandflies for high quality single cell rna sequencing |
| url | https://bio-protocol.org/en/bpdetail?id=5352&type=0 |
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