Wandering third instar larvae expressing Disc\RFP^{dsRNA.mCherry.UAS.VALIUM20} under the control of Scer\GAL4¹¹⁵¹ were harvested between 110 and 135 h after egg-laying. The notum and hinge regions of wing discs were dissected and cells were dissociated with collagenase and trypsin in Rinaldini solution.

Single-cell RNA-seq libraries were prepared using the DROP-seq method (Macosko et al., 2015).

RNA-seq libraries were sequenced on the Illumina NextSeq platform.

Dmel R6.32

Reads were processed using the Dropseq-tools v1.13 following the Drop-seq Core Computational Protocol v1.2. They were mapped to the Drosophila melanogaster reference genome BDGP6. Further analysis was performed using Seurat v3.0.0. For quality filtering, cells with less 200 genes or more than 2,500 genes were excluded, as well as cells with more than 10% of mitochondrial genes, more than 6% of HSP genes, or more than 1.5% of 28sRNA. For dimensionality reduction, principal component analysis was performed using the top 2,000 variable genes, and the first 30 principal components were selected as input for Uniform Manifold Approximation and Projection (UMAP) clustering.