scRNA-seq of posterior brain organoid morphogen screen
scRNA-seq of posterior brain organoid morphogen screen
After 5 weeks (36 days) in culture 1-4 organoids per condition were dissociated individually using CyBio FeliX liquid handler robot with thermoshaker. For dissociation we used a papain-based dissociation kit as for the time course. Each organoid was dissociated using 820uL of enzyme mix 1 and 12uL of enzyme mix 2. Then each individual single cell suspension has been followed by nuclei isolation, as described for the time course (lysis time 2.5 minutes). Nuclei fixation and permeabilization procedures were performed according to the manufacturer specification (ParseBiosciences, nuclei fixation kit v2.1.2, WN400).  Brain organoids were generated from the WTC cell line. For the morphogen screen experiment protocol 113 was used as a baseline and also serves as a control. Organoids were plated in ultra low-attachment plates (Corning, CLS7007), 5 organoids per condition. On day 15 they were transferred in 6-well plates, placing all 5 organoids per condition in one well and kept on shaker, as in the original protocol.  The majority of morphogens were provided between day 8 and 14 (Supplementary Table 5), always using base medium as indicated in the original protocol. Exception being SHH, which was provided from day 1 to day 7, and CHIR, from day 5 to day 14, as in the original protocol; insulin, being provided from day 1 to 14 as in the published cerebellum protocol18; FGF-19 - from day 15 onwards, as it has been known to promote neural differentiation. We have used the following morphogens: SHH (R&D Systems, 1845-SH-025/CF), CHIR-99021 (Tocirs, 4423), insulin (Sigma, I9278), R-Spondin-3 (Peprotech, 120-44), R-Spondin-2 (Peprotech, 120-43), retinoic acid (Sigma-Aldrich, R2625), FGF-8 (STEMCELL Technologies, 78204), FGF-19 (Peprotech, 100-32), FGF-17 (Peprotech, 100-27), FGF-2 (Mitenyli Biotech, 130093840), BMP4 (Mitenyli Biotech, 130-111-167). Then collected samples were processed for highly multiplexed single-nucleus RNA sequencing (snRNA-seq) using a split-pool combinatorial barcoding kit (ParseBiosciences, WT Mega kit v2, dual-index version RX200). Then collected samples were processed for highly multiplexed single-nucleus RNA sequencing (snRNA-seq) using a split-pool combinatorial barcoding kit (ParseBiosciences, WT Mega kit v2, dual-index version RX200).