X10 system

The w¹¹¹⁸ flies were used to harvest tracheal cells. Briefly, 100 tracheae of 0 h APF pupae were dissected in cold Grace medium supplemented with 2.5% fly extract, 1 mM PMSF, and EDTA-free protease inhibitor cocktail (Roche) within 1 h and digested in 1 mg/ml Elastase (Sigma, #E0258) solution at 25 °C for 1 h. Dissociated cells were pelleted at 400 x g for 20 min, resuspended in PBS with 0.2% BSA, filtered with 70 mm filters (BD Falcon), and sorted using a FACS Aria III sorter (BD Biosciences). TC20 automated cell counter (BIO-RAD) was used to assess cell number and live/dead cell ratio. About 8000 live cells (ratio of live cells in the suspension >90%) were obtained for 10X genomics library preparation following the manufacturer’s manual. Briefly, single cells and GemCode Gel Beads were encapsulated into droplets by using the GemCode Single-Cell Platform, and within-droplet cell lysis and barcoded reverse transcription reaction were carried out, which generated ~100 ng of cDNA after 12 cycles of reverse transcription. Following standard library preparation, next-generation deep sequencing was then carried out on the Illumina X10 system (Novogene).

Cells were administered the vehicle control and S34F mutants in triplicate for 96 h, and total RNA was extracted with TRIzol reagent (Invitrogen, CA, USA) following the vendor’s process. RNA sequencing was performed on the Illumina X10 system of LC Sciences (USA). R software package was adopted for processing the DEGs. Pathway enrichment analyses of the dysregulated genes were also carried out. Our study employed the Gene Set Enrichment Analysis (GSEA) online database to identify the interactions of DEGs in the apoptosis-related cluster.