scRNAseq datasets of human brain organoids generated for the manuscript \"Morphodynamics of human early brain organoids
scRNAseq datasets of human brain organoids generated for the manuscript \"Morphodynamics of human early brain organoids
For all experiments, single-cell suspensions were generated by dissociation of the organoids with a papain-based neural dissociation kit (Miltenyi Biotec, 130-092-628). Briefly, organoids were washed three times with HBSS without Ca2+ and Mg2+ (STEMCELL Technologies, 37250). Prewarmed papain solution (1-2 ml) was added to the organoids and incubated for 15 min at 37°C. The tissue pieces were triturated 5–10 times with 1000 µl wide-bore and then P1000 pipette tips. The tissue pieces were incubated twice for 10 min at 37 °C with additional trituration steps in between and after with P200 and P1000 pipette tips. Cells were filtered consecutively with a 30 or 40µm filter, centrifuged at 300g for 5 min and resuspended in cold PBS. The viability and cell count for the single cell suspensions were assessed using a Trypan Blue assay on the automated cell counter Countess (Thermo Fisher Scientific).  For the experiments with YAP activator (Fig. 6e,f, Extended Fig. 10h-l), organoids were generated using the unlabelled WTC-11 parent iPSC line. For the scRNAseq (Fig. 6e,f) organoids were cultured as described above with addition of 1:1000 DMSO in neural induction medium to control organoids on day 5 and 10µM Py-60 (MedChem Express, HY-141644) to organoids on day 5 or day 7. Media with fresh inhibitor or control media were exchanged every other day till dissociation and sequencing on day 10. For the scRNAseq (Extended Figure 9c-f; Extended Figure 10c-g), EBs were generated from 500 cells in mTSR+ (1:200 Rocki and 1:200 Pen-strep), per well of a 96 well plate and centrifuged at 200g for 5 min to generate embryoid bodies (EBs). Fresh mTSR+ with 1:200 ROCKi and 1:200 Pen-strep was exchanged on day 2 and day 4. Fresh Neural induction media was supplied on day 6 and exchanged every other day, followed by differentiation media - VitA on day 10 and differentiation media + VitA on Day 15. Organoids were given 2% matrigel, or 10µM Py-60 with 2% matrigel or no matrix on day 10. All media compositions were exchanged on day 13 followed by dissociation and sequencing on day 16. For scRNA-seq, cells were resuspended to a final concentration after counting and viability checking that enabled targeting 8,000 cells and, in case the cell numbers were not sufficient, all cells were loaded. The scRNA-seq libraries were generated using the Chromium Single Cell 3′ V3 Library & Gel Bead Kit. Single-cell encapsulation and library preparation were performed according to the manufacturer's protocol. Single-cell encapsulation and library preparation were performed according to the manufacturer's protocol.