GSM5057672: GER047_Axo_BL11dpa; Ambystoma mexicanum; RNA-Seq
Single-cell suspensions were obtained by chopping the tissues into <1mm cubes and immediately transferring the cubes to a tube of digesting solution (0.13WU/mL Liberase in 0.7x PBS). The enzymatic digestion was placed on a rotating wheel at room temperature for 45 minutes and then the cells were mechanically dissociated by pipetting 10-15 times with a P1000 pipet tip. Enzymatic reactions were stopped by adding high-serum culture medium (HS-AMEM, 125ml MEM, 20ml heat inactivated A1 FCS, 2ml insulin, 2mL glutamine, 2mL Pen/Strep, 50mL ddH2O), and samples were filtered sequentially through 70 μm and 30 μm MACS® SmartStrainers (Miltenyi Biotec, 130-098-458 and 130-098-462). Dissociated cells were pelleted using 300x rcf centrifugation for 5 minutes at room temperature, then washed twice with 0.7x PBS before resuspension in serum-free medium (114.3ml F12:DMEM+Glutmax, 1.5ml Sodium pyruvate, 1.5ml B27 supplement, 1.5ml MEM NEAA, 1.5ml insulin, 1.5ml Pen/Strep, 28.2mL ddH2O) on ice. Single-cell cDNA and sequencing libraries were generated following the manufacturer recommendations (10X Genomics, 3' library kit, v3 chemistry). The quality of the cDNA and resulting sequencing libraries were checked by either bioanalyzer or fragment analyzer.