GSM6311611: organoid 1; Homo sapiens; RNA-Seq
Individual cerebral organoids were dissociated using the Worthington Papain Dissociation System (Worthington). Organoids  were cut into pieces with a sterile scalpel blade and washed twice in PBS prior to dissociation. Incubation in papain/DNaseI solution was performed at 37 ºC and 5% CO2 with occasional agitation. After the first 30 minutes of incubation, organoids were triturated 10 times with a fire-polished glass Pasteur pipette, followed by further incubation and trituration every 15 minutes until dissociation was complete. The total incubation time in papain/DNaseI solution was 90 minutes. After adding Inhibitor Solution and DNaseI in EBSS, the dissociated cells were pelleted by centrifugation for 3 minutes at 300 g. The cells were resuspended in 500 µl ice-cold PBS/0.01% BSA using a wide-bore plastic pipette tip and passed through a 35 µm cell strainer. An aliquot of the single-cell suspension was mixed with Trypan blue and analyzed for viability and cell concentration on a Countess II FL Automated Cell Counter (ThermoFisher). cells were diluted to 110 cells/µl in PBS/0.01% BSA. Drop-seq was performed as described in Macosko et al. (Macosko et al., 2015; DOI: 10.1016/j.cell.2015.05.002). Briefly cells and barcoded beads (Chemgenes, 132 beads/ul in lysis buffer) were run on an aquapel-treated microfluidic drop-seq device (FlowJEM) for co-encapsulation in nanoliter-sized droplets. After droplet-breakage, reverse transcription and Exonuclease I treatment, beads were counted, and 2,500 beads were apportioned per PCR tube for cDNA amplification. The amplified cDNA libraries were purified using SPRI beads (Beckman Coulter) and quantified on a Fragment Analyzer (Agilent). Tagmentations were performed using the Illumina Nextera XT kit (Illumina), and the resulting libraries were purified in two consecutive rounds of SPRI beads-based size selection (0.6x beads to sample ratio followed by 1x beads to sample ratio). The size and concentration of the final libraries were measured on a Fragment Analyzer and a Qubit Fluorometer (ThermoFisher), respectively.