We collected 35-50 hours old embryos of Tribolium and transferred them into a washing basket. We washed the embryos several times in Milli-Q water, and subsequently dechorionated the embryos with 5% bleach for 3 minutes under constant shaking. We washed the embryos extensively with Milli-Q water. Henceforth, we kept all solutions, tubes, and embryos on ice. Using a paintbrush, we transferred the embryos into a 2 mL dounce homogenizer prefilled with 1 mL of Schneider's medium mixed with 1% BSA. We then mechanically dissociated the embryos by using approximately 15 strokes of the pestle A. We decanted the homogenate into a 1.5 mL vial and centrifuged it at 50g for 3 minutes at 4 °C to remove chunks of tissue. We decanted the supernatant into a new 1.5 mL vial and centrifuged it at 300g for three minutes at 4 °C. We discarded the supernatant and resuspended the pellet in one milliliter of Schneider's medium mixed with 1% BSA. We filtered the suspension with a 20 μm cell strainer coated with a thin layer of 1% BSA and centrifuged again for three minutes at 4°C. Finally, the cell pellet was resuspended in 50 μL of cold Schneider's medium mixed with 1% BSA supplemented with 0.8% protector RNase-inhibitor and 1x Protease Inhibitor Cocktail. Using a cell sorter SH800S cells were sorted for one hour. To set the gating parameters for FACS, we used unstained cell suspensions from wild-type Tribolium embryos as a negative control and DAPI stained wild-type embryos to set the gating for live versus dead cells. Once the gating parameters were set, we started by analyzing all events in our samples stained with DAPI using the forward scatter (FSC-A) and the side scatter (BSC-A) without gating. We then gated using forward scatter (FSC-A) and side scatter (FSC-W) to discard doublets. We then created a third plot using forward scatter (FSC-A) and fluorescent (DAPI), selecting DAPI-negative cells. Finally, these selected events were plotted again using phycoerythrin (PE) versus fluorescent (GFP) to exclude auto-fluorescent cells and to identify a small cell population of highly GFP-fluorescent cells. We sorted the highly GFP-fluorescent cells into a centrifuge tube coated and prefilled with 50 μL of ice-cold Schneider's medium mixed with 1% BSA. We generated single-cell RNA libraries using the Chromium Next GEM Single Cell 3ʹ Kit v3.1 and a 10x Chromium controller (10x Genomics) following the manufacturer's protocol instructions. The resulting cDNA libraries were quantified using a bioanalyzer and sequenced in an Illumina NextSeq 2000 Sequencing Systems (Illumina, Inc.) to an average depth of 50,000 reads per cell.