title: RNA-seq of microglia and monocyte-derived cells in Nrf2 knockout and wild-type mice after induced intracerebral hemorrhage or sham treatment
design: RNA-seq of microglia and monocyte-derived cells in Nrf2 knockout and wild-type mice after induced intracerebral hemorrhage or sham treatment
library_construction_protocol: 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).