GSM8863196: sorted mCherry+ tenocytes from uninjected 48 hpf Tg(scxa:mCherry) zebrafish embryos replicate 6; Danio rerio; RNA-Seq
For 48 hpf bulk RNA-seq alone transgenic Tg(scxa:mCherry) embryos, BTX-injected or un-injected siblings, were dissociated using Subtilisin A cold-active protease in a stock solution consisting of: 5 ul of 1M CaCl2, 100 ul of protease stock solution (100mg of Bacillus licheniformis protease (Sigma P5380) solubilized in 1 ml of Ca and Mg free PBS), 889ul of PBS, 1 ul of 0.5M EDTA (Sigma E5134) and 5ul of DNAse I  (Roche 10104159001) stock (25U/ul in PBS, stored at -80C) adapted from (O'Flanagan et al., 2019). Embryos were triturated once every 2 min for 15 seconds using a wide bore 1 ml pipette. Every 15 min, tissue solution as checked under dissecting scope to verify dissociation. Full dissociation took ~30 min per samples, and samples were subsequently run through a 40 mm filter to separate dissociated cells from clumps of aggregate undissociated tissue/ECM and washed with 10 ml of PBS/BSA (0.01% BSA in PBS, made fresh on the day of dissociation) and transferred to a 15 ml conical tube. Cells were centrifuged at 600g for 5 min at 4°C, supernatant is discarded, and cells are resuspended in 1 ml of ice-cold PBS/BSA before being placed on ice. High expressing mCherry+ cells were gated and sorted on a Bio-Rad FACS Aria II cell sorter. Clontech SMARTer Stranded Total RNA-Seq v3 Kit