GSM8995777: CorticalSurfaceAssociated_scRNAseq_3; Mus musculus; RNA-Seq
For single cell isolation the entire dorsal and lateral V-SVZ were microdissected out. Tissues were finely chopped and incubated in Hibernate A minus calcium medium (Brain Bits) supplemented with 2% B27, 20 units/mL papain, and 0.005% DNaseI for 30 minutes at 37°C in the dark. This enzymatic incubation solution also contained DRAQ5, a membrane permeable dye to stain for live cells (at 5 μM or 3000- fold dilution, Thermo Fisher). After enzymatic treatment, the tissue suspension was centrifuged at 300 x g for 5 minutes and resuspended in HBSS/0.025% BSA. A cell suspension was produced via manual trituration using blunt needles of sequentially decreasing diameter. The resultant cell suspension was filtered through a 40 μm strainer and labeled with 1 μg/mL propidium iodide (PI, Abcam). Viable cells were collected using flow cytometry sorting for propidium iodide-negative and DRAQ5-positive cells. Libraries were prepared according to the manufacturer's instructions (10X Genomics) using the Chromium Single Cell 3' Library and Gel Bead Kit.