GSM7871949: WT 5.8 mm; Danio rerio; RNA-Seq
Fish were euthanized and transferred to ice-cold PBS. In <2 min, we cut off the head just above the pectoral fins, and removed the eyes and brain to enrich for musculoskeletal tissue.The remaining skull was rinsed 2X in PBS, and transferred to a dissociation buffer containing 0.25% trypsin-EDTA and 10mg/mL collagenase at 30°. Alternating between pipetting and incubation at 30°, the tissue was fully dissociated, again in <2 min. Digestion was stopped by addition of DMEM w/ 10% BSA. After centrifugation at 700g for 5min, cells were resuspended in PBS, centrifuged again to wash, and the final pellet resuspended in 100μL of FACS buffer (1x PBS, 2% BSA and 1mM EDTA). Single cells were isolated by filtration through a 40μm nylon mesh. Cells were loaded onto 10X Genomics microfluidic chips and processed for scRNAseq according to manufacturer's instructions.