GSM9060723: Mouse brain endothelial cells, contralateral hemisphere, PF-429242 treated MCAO; Mus musculus; RNA-Seq
For FACS sorting, cell suspensions were obtained upon digesting brain tissue in 2 mg/ml Dispase II (D4693, Sigma-Aldrich), 2 mg/mL Collagenase IV (#1710401, Thermo Fisher Scientific, Zurich, Switzerland) and 2 mM CaCl2 PBS (40 min at 37°C). The suspension was filtered through 100/70/40 μm cell strainers (#431751, Corning, New York, USA) and cells were then centrifuged 300 g for 7 min, and resuspended in 9 ml PBS, followed by another round of centrifugation. Cell pellets were resuspended in 3 mL of ACK hemolytic buffer. To stop the reaction, 30 mL of ice-cold PBS was added and centrifuged at 300 g for 7 min. Pellets were resuspended in PBS + 1% BSA. For FACS sorting, cells were stained with anti-CD31 and anti-CD45 (see Table 3). Following washes with PBS + 1% BSA the suspension was passed through a 35 μm cell strainer of a FACS sorting tube (#352235, Corning). Cells were sorted by a FACS Aria III (648282D2, BD Bioscience) sorter using the four-way purity sorting mode.  Live events were acquired and analyzed using Flowjo 10.0.6 software (Tree Star Inc.).  FACS-sorted endothelial cells were resuspended at a 1,000 cells/μL. scRNA-seq libraries were obtained following the 10x Genomics recommended protocol using the reagents included in the Chromium Single Cell v3.3 Reagent Kit. Libraries were sequenced on a NextSeq 500 (Illumina, Inc., San Diego, CA, USA) at the Princess Marget Genomics Center (University Health Network, Toronto, ON, Canada), aiming at 50k reads per cell.