Protocol for cell-type-specific single-cell labeling and manipulation in Drosophila using a sparse driver system

Here, we present a protocol for cell-type-specific single-cell labeling and manipulation in Drosophila using a sparse driver system. We describe steps for generating constructs and fly lines, titrating heat-shocked durations for precise temporal control and desired sparsity, and co-expressing multiple transgenes for experiments. We demonstrate that this generalizable toolkit enables tunable sparsity, multi-color staining, single-cell trans-synaptic tracing, single-cell manipulation, and cell-autonomous gene function analysis. For complete details on the use and execution of this protocol, please refer to Xu et al.¹.