GSM8787242: D19_0G_GEX; Homo sapiens; RNA-Seq
COs for single-cell RNA sequencing were processed with the Brain Tumour Dissociation Kit (P) (Miltenyi Biotec,130-095-942) following the manufacturer's protocol and using the gentle MACS dissociator.  24 hrs post-irradiation, 3-5 pooled COs 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 gentle MACS 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. Library preparation was performed at the Single Cell Genomics Facility (UCL, Cancer Institute) using the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics, 1000268) along with the Chromium Next GEM Chip G Single Cell Kit (10X Genomics, 1000120) according to the manufacturer's instructions. Pooled samples libraries were sequenced with lane sequencing on Novaseq 6000 S4 (Novogene).