GSM7437213: sham [scRNA-Seq]; Mus musculus; RNA-Seq
The Plp1/tdTomato mice were deeply anesthetized with pentobarbital (PTB) (100 mg/kg, i.p.), and perfused transcardially with a cold (4–8°C) isolation buffer containing (in mM): NaCl 136.0, KCl 5.4, HEPES 10.0, glucose 5.5, osmolality 290±3 mOsmol/kg. To isolate the control and ischemic areas, the brain was sliced into 600 µm coronal sections using a vibrating microtome Leica VT1200S (Leica Microsystems). The collected tissue was incubated with continuous shaking at 37°C for 45 min in 1 ml of papain solution (20 U/ml) and 0.2 ml DNase (both from Worthington) prepared in isolation buffer. After papain treatment, the tissue was mechanically dissociated by gentle trituration using a 1 ml pipette. The dissociated cells were layered on top of 5 ml of ovomucoid inhibitor solution (Worthington) and harvested by centrifugation (70 x g for 6 min). This method routinely yielded ~2 x 10^6 cells per mouse brain. Cell aggregates were removed by filtering with 70 µm cell strainers (Becton Dickinson). Three animals per condition were pooled in the preparation of cell suspension. After obtaining an aliquot for FACS, the cell suspension was spin down, concentrated, and used for library preparation. Single cell suspension from Plp1/tdTomato mice were sorted using fluorescent activated cell sorting (FACS; BD Influx). The flow cytometer was manually calibrated to deposit a single cell in the center of collection tube. Hoechst 33258 (Life Technologies) was added to the suspension of cells to check viability. 3000 Plp1+ cells from each group (control, D1, D3, D7) were collected into 96-well plates (Life Technologies) coated with BSA (ThermoFisher Scientific) and containing 5 μl of DMEM (ThermoFisher Scientific) and 15% FBS (HyClone). Single-cell RNA-Seq (scRNA-Seq) libraries were prepared using Chromium Next GEM Single Cell 3' Kit v3.1 (1000268, 10X Genomics) according to the manufacturer's protocol.