title: GSM8440146: dorsal forebrain organoids day 90; Homo sapiens; RNA-Seq
library_construction_protocol: 5 organoids of each condition at day 90 were pooled together and washed three times with HBSS without Ca2+ and Mg2+ (STEMCELL Technologies, 37250). Single-cell suspensions were acquired via incubation with a papain-based dissociation (Miltenyi Biotec, 130-092-628). Prewarmed papain containing mix 1 (2 ml) was added to the organoids and incubated for 15-20 min at 37 °C with gentle agitation every 5 minutes. After triturating 5–10 times with 1,000 µl wide-bore, enzyme mix 2 was added and incubated for 10 minutes at 37 °C twice with P1000 and P200 pipette tips trituration steps. Cells were washed twice with PBS + 0.2% BSA and filtered through a 40 µm cell strainer. Viability and cell count were assessed using a Trypan Blue assay on the automated cell counter Countess (Thermo Fisher Scientific). Cells were centrifuged at 300g at 4℃ for 5mins and resuspended in ice-cold PBS + 0.2% BSA at a cell density of 1000 cells/μL. Single-cell encapsulation and library preparation were performed according to the manufacturer's protocol. Approximately 12,000 cells in each channel were loaded onto a Chromium Single Cell 3′ Chip (10× Genomics, PN-120236) and processed through a Chromium controller to generate single-cell gel beads in emulsions (GEMs) according to the manufacturer's instructions. Captured libraries were prepared with the Chromium Single Cell 3′ Library & Gel Bead Kit v.2 (10x Genomics, PN-120237) and sequenced on an Illumina Novaseq with a paired-end 150 bp (PE150) reading strategy.