title: RNA-seq of endothelial cells, astrocytes and microglia in cell-type specific Nrf2 knockout mice, in a model of system inflammation
design: RNA-seq of endothelial cells, astrocytes and microglia in cell-type specific Nrf2 knockout mice, in a model of system inflammation
library_construction_protocol: Single brain cells were isolated using Adult Brain Dissociation kit (Miltenyie, 130-107-677, Germany) as per the manufacturer's instructions. The single brain cell suspension was then incubated with antibodies against CD31 (Biolegend, 102523, UK) CD11b (Biolegend, 101205, UK), ACSA2 (Miltenyie, 130-116-245, Germany), O4 (Miltenyie, 130-117-357, Germany), CD45 (Biolegend, 103125, Germany) for 15 minutes in ice, washed with PBS and then immediately FACS sorted into RNAprotect Cell Reagent (Qiagen, 76526, UK) under the gates of CD31+CD45-ACSA2- for brain endothelial cells, LY6G-CD11b+CD45low for microglia, ACSA+O4- for astrocytes. RNA extraction was carried out using the RNeasy Plus Micro Kit (Qiagen) as per the manufacturer's instructions.  RNA-seq libraries were prepared by Edinburgh Genomics using the Illumina TruSeq stranded mRNA-seq kit, according to the manufacturer's protocol (Illumina).