title: GSM8498073: Conventional midbrain organoids; Homo sapiens; RNA-Seq
library_construction_protocol: The organoids were dissociated into single-cell solutions using a commercially available papain-based dissociation (Neural tissue dissociation kit-P, Miltenyi Biotec) following the manufacturer's protocol with slight modification. The organoids were washed 3 times with HBSS (without Ca2+ and Mg2+, −/−) and cut into hemispheres with scissors. The cut hemispheres were then incubated in enzyme mix 1 (enzyme P) with 90 RPM orbital shaking for 15 minutes, followed by adding enzyme mix 2 (enzyme A) and incubation for another 30 minutes with orbital shaking. During the incubation, the organoid samples were triturated with a wide bore 1ml tip every 10 to 15 minutes. Then, the samples were filtered through a 30 μm strainer (Miltenyi Biotec) to remove the large aggregates and keep single cells. The single-cell solutions were centrifuged at 300 x g for 8 minutes at 4 oC, and resuspended to the desired concentration (1 million/ml) in the organoid medium without any growth factors, followed by 10X scRNA-seq. Library construction was performed using Next-GEM Chromium Single Cell 3' Library & Gel Bead Kit v3.1following the manufacturer's protocol.