RNA-seq of microglia and monocyte-derived cells in Nrf2 knockout and wild-type mice after induced intracerebral hemorrhage or sham treatment
RNA-seq of microglia and monocyte-derived cells in Nrf2 knockout and wild-type mice after induced intracerebral hemorrhage or sham treatment
We dissected brain tissue of mice killed by overdose of anaesthetic and placed brains in ice cold Dulbecco's phosphate buffered saline (DPBS; Invitrogen, 14287080). We removed the cerebellum and olfactory bulbs before performing mechanical and enzymatic dissociation using the gentleMACS Octo Dissociator, according to manufacturer's instructions (Miltenyi Biotech, 130-096-427). We removed debris and myelin using a cell strainer and gradient centrifugation (Miltenyi, 130-110-916). We applied red blood cell lysis solution (Miltenyi, 130-094-183). We estimated live cells isolated per brain using trypan blue exclusion (Sigma, T8154) and a haemocytometer. Next, we applied 1:100 rat anti-mouse Cd16/32 (BioLegend, 101302), Cd11b-FITC (1:200; BioLegend, 101206), Cd45-Pacific blue (1:50; BioLegend, 103126), ASCA-2-APC (1:200; Miltenyi, 130-116-245), Ly6g-PE (1:1000, BioLegend, 127607), O4-PE (1:200; Miltenyi, 130-117-357) and Draq7 (1:200; Invitrogen, D15105). FMO samples were also prepared. We isolated Microglia and MdCs using a FACSAria Il flow cytometer (Beckton Dickinson Immunocytometry Systems [BD]) on Purity precision, running BD FACSDiva (v6.13; Supplementary Figure 5). We determined the fraction of each cell type per sample using FCS Express 7 Research (v7.04.0014) and calculated the absolute number of each cell type per brain by taking the product of this and the total number of cells estimated earlier using a haemocytometer.  We isolated RNA from sorted cells immediately following sorting using RNeasy Plus Micro kits (Qiagen, 74034).  We produced cDNA for sequencing using the SMART-Seq V4 PLUS ultra-low RNA input kit (Clontech) and sequencing libraries using Nextera XT DNA library Preparation kit (Illumina).