A rapid, gentle and scalable method for dissociation and fluorescent sorting of imaginal disc cells for mRNA sequencing

Abstract

Dissociation of imaginal disc cells has been carried out previously to enable flow cytometry and cell sorting to analyze cell cycle progression, cell size, gene expression, and other aspects of imaginal tissues. However, the lengthy dissociation protocols employed may alter gene expression, cell behavior and overall viability. Here we describe a new rapid and gentle method of dissociating the cells of wing imaginal discs that significantly enhances cell viability and reduces the likelihood of gene expression changes. Furthermore, this method is scalable, enabling collection of large amounts of sample for high-throughput experiments without the need for data-distorting amplifications.

A Positive Feedback Loop Ensures Propagation of ROS Production and JNK Signaling ThroughoutDrosophilaTissue Regeneration

“…This work describes generation of a transcriptional profile of actively regenerating tissue, made possible by our genetically induced tissue ablation system (6) and our technical advances enabling isolation of sufficient numbers of blastema cells (41). Through analysis of the expression data, followed by functional validation of differentially expressed genes, we have discovered a key positive feedback loop that uses JNK-induced upregulation of the Duox-maturation factor encoded by mol to sustain ROS production, JNK signaling, and late regeneration.…”

“…Expression of GFP via this insertion occurs in the same cells that are immunostained with an anti-Nub antibody ( Fig 1C) (41,47). The wing primordium continues to express the nub-GFP after ablation and throughout different stages of regeneration ( Fig 1D-F).…”

“…Dissociation and fluorescence-activated cell sorting (FACS) of imaginal disc cells is a well-established but lengthy procedure that may affect gene expression and cell viability (49,50). We therefore optimized our cell dissociation process so that it was rapid and gentle, taking approximately 15 minutes, to minimize changes in transcription and loss of cell viability due to the manipulation of the tissue (41). We have previously confirmed the accuracy of the sorting by using qPCR to measure expression of pouch and non-pouch genes in the sorted cells (41).…”

“…We therefore optimized our cell dissociation process so that it was rapid and gentle, taking approximately 15 minutes, to minimize changes in transcription and loss of cell viability due to the manipulation of the tissue (41). We have previously confirmed the accuracy of the sorting by using qPCR to measure expression of pouch and non-pouch genes in the sorted cells (41). After using this protocol to dissociate and sort regeneration blastema cells and control wing pouch cells, mRNA was prepared and pooled such that each biological replicate produced sufficient mRNA for deep sequencing ( Fig 1H).…”

“…The ablated regenerating discs had the genotype nub-GFP/+; rn-Gal4, GAL80 ts , UAS-rpr /+, while the mock-ablated controls had the genotype nub-GFP/+; rn-Gal4, GAL80 ts /+. Cells were isolated for the transcriptional profile as previously described (41). Briefly, discs were dissected using teams of 4 researchers dissecting simultaneously to maximize the number of discs obtained per sample.…”