GSM7776164: x19shM7B; Homo sapiens; RNA-Seq
Selected mice for scRNAseq were culled by neck dislocation and the brains were dissected and placed into HBSS (Gibco, 14175095) in petri dishes. Brains were cut into small pieces with a surgical blade and then dissociated into single cell suspension using the Brain Tumour Dissociation Kit (P) (Miltenyi Biotec,130-095-942) following the manufacturer's protocol. Cells were centrifuged at 300g x for 10 minutes, followed by cell debris removal using the Debris Removal Solution (Miltenyi Biotec, 130-109-398) as per protocol instructions. Mouse cells were removed using the Mouse Cell Depletion Kit (Miltenyi Biotec, 130-104-694) as per manufacturer instructions with minor adaptations. Cells were centrifugeds and manually counted. 30000 cells were isolated, centrifuged again at 300g x 5 minutes and re-suspended in 0.04% BSA in PBS at a concentration of 1000 cells/µl. COs for single-cell RNA sequencing were processed with the same Neural Tissue Dissociation Kit (P) using the gentle MACS dissociator. 3-5 pooled day70 COs derived from gene-edited EPSC were harvested and washed with D-PBS without calcium and magnesium. The organoids were then transferred into a C-Tube containing the pre-heated enzyme mix 1 followed by the gentleMACS Program m_brain_01, 37⁰C incubations under slow continuous rotation, gentleMACS Program m_brain_02, addition of enzyme mix 2 and gentleMACS Program m_brain_03. Single-cell suspension was passed through a 70µm strainer and centrifuged at 300g x for 10 minutes. CO cells were then resuspended in 0.04% BSA in DPBS, and viability and cell number were assessed with the Countess® II FL Automated Cell Counter using trypan blue. 100,000 cells were aliquoted into a new tube in 100µl to achieve a cell density of 1000 cells/µl as required by the 10X Genomics Single-cell RNA sequencing protocol, followed by a final count to ensure the correct number of cells. The Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics, 1000268) was used along with the Chromium Next GEM Chip G Single Cell Kit (10X Genomics, 1000120).