GSM9066342: WT; Danio rerio; RNA-Seq
10 guts from 5 dpf larvae were dissected and placed in 200 μL of a dissociation cocktail (1 mg/mL fresh collagenase A, 40 μg/mL proteinase K, and 0.25% trypsin in PBS) at 37°C for 30 minutes. 50 guts were dissected per condition for a total of 5 replicate tubes per condition. Cells were centrifuged at 300 RCF for 15 minutes at 4°C. The pellet was resuspended in 200 μL PBS with 0.04% non-acetylated BSA. Replicates were pooled and filtered through a 40 μm cell strainer (Fisherbrand, #22363547). Samples were centrifuged at 300 RCF for 15 minutes at 4°C and resuspended with in 50 ul of PBS with 0.04% non-acetylated BSA. To isolate live cells, an OptiPrep™ Density Gradient Medium (Sigma, #D1556-250ML) was used. A 40% (w/v) iodixanol working solution was prepared by mixing 2 volumes of OptiPrep™ with 1 volume of 0.04% BSA in 1X PBS/DEPC-treated water. This working solution was used to create a 22% (w/v) iodixanol solution in the same buffer. The cell suspension was mixed with one volume of the working solution and 0.45 volume of the cell suspension via gentle inversion, transferred to a 15 mL conical tube, and topped up to 6 mL with the working solution. This solution was overlaid with 3 mL of the 22% iodixanol, and then with 0.5 mL of PBS with 0.04% BSA. Samples were centrifuged at 800 RCF for 20 minutes at 20°C. Viable cells were collected from the top interface, diluted in PBS with 0.04% BSA, and centrifuged at 300 RCF for 10 minutes at 4°C. The supernatant was discarded, and the cells were resuspended in PBS with 0.04% non-acetylated BSA to reach the desired concentration. Data analysis was performed using the Seurat package in R (Hao et al, 2024). Cells were loaded onto a 10× Chromium instrument (10× Genomics) and libraries were prepared using the Single Cell 3′ Reagent Kit (V2 chemistry) (10× Genomics) according to the manufacturer's instructions. The sequencing coverage was approximately 100,000 reads per cell