GSM8660172: WA09_invitro_GPC_CD140+_2; Homo sapiens; RNA-Seq
For invitro samples unsorted PSC and GPC stage cells were incubated in Accutase (Millipore Sigma Cat. A6964) diluted 50% in DPBS for 6 minutes at 37 degrees. Cells were resuspended in DMEM and filtered for FACS. . Cells were selected based on forward and side scatter in addition to negative gating of 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, D1306) staining to remove dead cells and debri (1 µg ml−1). Cells were then captured for scRNA-seq on a 10X Genomics chromium controller. For Invivo samples the mouse brains were removed and immersed in ice-cold HBSS and their corpora callosa dissected and chopped into small pieces. This tissue was then incubated in a papain/DNase dissociation solution at 37 °C for 50 min. Papain was inactivated with the addition of ovomucoid dissolved in EBSS. A single-cell suspension was then achieved via repeated pipetting. Cells were pelleted, resuspended into MEM, and filtered for FACS. Cells were selected based on forward and side scatter in addition to negative gating of 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, D1306) staining to remove dead cells and debri (1 µg ml−1). Cells were then captured for scRNA-seq on a 10X Genomics chromium controller. Libraries were constructed according to manufacturers instructions for either single cell 3' v2 or single cell 3' v3.1 from 10X genomics as noted in the metadata table.