GSM8774088: GFAPsh-SA6-iCre-miR124T, 50 days post injection n2; Mus musculus; RNA-Seq
Brains were harvested into cold 1X PBS 7 and 50 days post AAV injection. Using a brain slicer, brains were cut into coronal sections and the injected region in the cortex was microdissected. Dissected tissue was dissociated into single cell suspension according to the manufacturer's instructions using Worthington Papain System kit (Worthington, LK003150). Using Dead cell removal kit (130-090-101, Miltenyi Biotec), dead cells were removed following the manufacturer's instructions. Trypan blue staining was used to determine cell viability, and samples with 90% viable cells were processed for analysis. Single-cell libraries were prepared using the Chromium Next GEM Single Cell 5' Reagent Kit v2 (10X genomics, Cat # 1000263), following the manufacturer's protocol. Briefly, cell suspensions were prepared at a concentration of 1 × 10⁶ cells/mL, and 16500 cells were loaded into each lane of a Chromium GEM Chip K for an expected recovery of 10,000 cells. Gel Bead-in-Emulsions (GEMs) were generated using the Chromium Controller (Wang Lab, Biological Sciences Platform of Sunnybrook Research Institute) followed by cDNA generation and amplification. For each sample, a total of 50 ng of cDNA was used to generate libraries according to the manufacturer's instructions. Equal molar quantities of each library were sequenced on the Illumina NovaSeq 6000 platform using the S4 flow cell system (The Centre for Applied Genomics, The Hospital for Sick Children, Toronto).