GSM5220257: 5d after tMCAO 1; Mus musculus; RNA-Seq
Ischemic hemispheres were perfused with ice-cold saline and freshly harvested at 5 or 14 days after tMCAO. Single cell suspensions were prepared for flow cytometry. Single cells were incubated with anti-CD45-PE-Cy5 antibody for 30 minutes on ice at 4 ℃ in the dark. The CD45high cells were sorted by FACSAriaTM (BD Bioscience). Single live cells were separated into Gel bead in emulsion (GEM)s formed by oil micro-droplets by the Chromium instrument (10X Genomics) using Single Cell 3' v2 reagents. Each GEM contains a gel bead and a cell. A greater than 1000-fold excess of partitions compared to cells assures that most GEMs were only one cell/GEM. The reaction mixture/emulsion with captured and barcoded mRNAs were removed from the Chromium instrument followed by reverse transcription. The cDNA samples were fragmented and amplified per 10X protocol. The libraries were then purified, quantified, and sequenced on an Illumina HiSeq.