Posterior brain organoids multi-omics atlas
Posterior brain organoids multi-omics atlas
Organoids of the three different cell lines were pooled on the basis of size and dissociated together, and the cell lines were later demultiplexed on the basis of the single-nucleotide polymorphism information. Multiple organoids of each line were pooled together to obtain a sufficient number of cells. If needed, on the later time points organoids were cut in halves and washed three times with HBSS without Ca2+ and Mg2+ (STEMCELL Technologies, 37250). Tissue dissociation to single cell suspension was done with a papain-based dissociation kit (Miltenyi Biotec, 130-092-628). Prewarmed papain solution (2 ml) was added to the organoids and incubated for 15 min at 37 °C. This was followed by enzyme mix A addition and then tissue pieces were triturated 5–10 times with 1,000 μl wide-bore and P1000 pipette tips. After that the tissue pieces were incubated twice for 10 min at 37 °C with trituration steps with P1000 and P200 pipette tips. After dissociation cells were filtered with 30 um filters and centrifuged. Before nuclei isolation, 100,000 cells were washed twice with 50 μL PBS containing 0.04% BSA. To isolate nuclei from the ready single-cell suspension, cells were resuspended in 50 μL lysis buffer (10 mM Tris-Cl pH7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20, 1 mM DTT, 1U/μL RNase inhibitor (Roche Protector RNase-Inhibitor), 0.1% NP-40, 0.01% Digitonin (Invitrogen, BN2006)) and incubated for 3 minutes on ice, neutralized by adding 50 uL wash buffer (10 mM Tris-Cl pH7.4, 3 mM NaCl, 10 mM MgCl2, 1% BSA, 0.1% Tween-20, 1 mM DTT, 1U/μL RNase inhibitor).  Brain organoids were generated from three different stem cell lines (WTC, WIBJ2, HOIK1) simultaneously. Brain organoids of the same batch were dissociated at multiple time points of the course of brain organoids development: neural induction (day 7) and neural differentiation and maturation (days 15, 30, 60, 90, 120).  Single-nuclei suspension- generation and library preparation were performed according to the 10x Chromium Single Cell Multiome ATAC + Gene Expression kit protocol. Single-nuclei suspension- generation and library preparation were performed according to the 10x Chromium Single Cell Multiome ATAC + Gene Expression kit protocol.