Background
Recent technological advances opened the opportunity to simultaneously study gene expression for thousands of individual cells on a genome-wide scale. The experimental accessibility of such single-cell RNA sequencing (scRNAseq) approaches already allowed gaining insights into the cell type composition of heterogeneous tissue samples of animal model systems and emerging models alike. A major prerequisite for a successful application of the method is the dissociation of complex tissue into individual cells, which often requires large amounts of input material and harsh mechanical, chemical and temperature conditions. However, the availability of tissue material may be limited for small animals, specific organs, certain developmental stages or if samples need to be acquired from collected specimens. Therefore, we evaluated different dissociation protocols to obtain single-cells from small tissue samples of Drosophila melanogaster eye-antennal imaginal discs.
Results
We show that a combination of mechanical and chemical dissociation resulted in sufficient high-quality cells. As an alternative, we tested protocols for the isolation of single nuclei, which turned out to be highly efficient for fresh and for frozen tissue samples. Eventually, we performed scRNAseq and single-nuclei RNA sequencing (snRNAseq) to show that the best protocols for both methods successfully identified relevant cell types. However, snRNAseq resulted in less artificial gene expression that is caused by rather harsh dissociation conditions needed to obtain single cells for scRNAseq.
Conclusion
We present two dissociation protocols that allow isolating single cells and single nuclei, respectively, from low input material. Both protocols resulted in extraction of high-quality RNA for subsequent scRNAseq or snRNAseq applications. If tissue availability is limited, we recommend the snRNAseq procedure of fresh or frozen tissue samples as it is perfectly suited to obtain thorough insights into cellular diversity of complex tissue.