GSM9514182: scRNA-seq of mouse microglia, MCAO Day 28, ASO control; Mus musculus; RNA-Seq
For microglia samples: Sham-operated mice or mice after MCAO were transcardially perfused with saline, and then the forebrain was removed. After homogenization with RPMI-1640, 1 mg ml–1 collagenase (Sigma-Aldrich) and 50 mg ml–1 DNase I (Sigma-Aldrich) were added to the homogenates. Brain homogenates were digested for 25 min and subjected to Percoll (GE Healthcare) gradient centrifugation. From the interlayer between 37% and 70% Percoll, cells were isolated and stained with anti-CD45 FITC (30-F11, eBioscience), anti-CD11b PerCP-Cy5.5 (M1/70, eBioscience), and anti-CD31 PE/Cyanine7 (390, eBioscience). CD45(int)CD11b(int) cells were sorted using a FACSAria III cell sorter (BD/Thermo Fisher Scientific). For whole brain cell samples: Brain cells including neurons were isolated from adult ischemic brain according to the protocol established in the Allen Institute (Tasic et al., Nature 2018); the detailed experimental method was described previously (Nakamura et al., Neuron 2023). Brain cells including neurons were isolated from adult ischemic brain according to the protocol established in the Allen Institute (Tasic et al., Nature 2018); the detailed experimental method was described previously (Nakamura et al., Neuron 2023). Single CD45(int)CD11b(int) cells or brain cells were loaded onto chromium chips with a capture target of 2,000 to 10,000 cells per sample. Libraries were prepared using Chromium Next GEM Single Cell 5′ Reagent Kits v2 (10x Genomics) for CD45(int)CD11b(int) cells or Chromium Next GEM Single-cell 3′ Reagent Kits v3.1 (10x Genomics) for brain cells, according to the manufacturer's protocol. On a DNBSEQ-G400 sequencer (MGI), obtained libraries were sequenced with a targeted sequencing depth of more than 50,000 read pairs per cell.