GSM8003385: mouse inner ear organoid, day 21, rep1; Mus musculus; RNA-Seq
For DIV3 and DIV4 time points, organoids were washed once in 1X PBS, incubated in Accutase dissociation reagent for 3 minutes at 37°C, and dissociated by manual trituration with a P200 pipette tip. This step was repeated twice. DIV8, DIV16 and DIV21 organoids were first incubated for 20 minutes in 0.5mg/mL Thermolysin at 37°C before incubation in Accutase for 3 minutes. Organoids were then mechanically dissociated using a 23G blunt ended needle connected to a sterile 1mL syringe, returned to the incubator for an additional 3 minutes, and dissociated a second time. Cells from all time points were then collected in 1mL 1X PBS, spun down at 4°C at 400xg for 10 minutes, and resuspended in 400μL of DMEM/F12+10%FBS. Cell clumps were removed by filtering through a 35μm strainer cap. Cells were then placed on ice and brought immediately to the Stanford Functional Genomics Core for 10X Genomics scRNA-seq capture and sequencing. 10X Genomics Single Cell 3' v3