DNA methylome and transcriptome sequencing to define pathways involved in the activation of microglia

Microglia cells are macrophages which reside in the brain, and become activated during the inflammatory processes involved in neurodegenerative and autoimmune disorders. An understanding of the molecular processes involved in this activation process could lead to the development of new neuroprotective therapies. To reveal molecular mechanisms involved in microglia activation, we performed RNA-seq and MBD-isolated genome sequencing (MiGS or MBD-seq) on both naïve and peak microglia derived from the mouse experimental autoimmune encephalomyelitis (EAE) model. The EAE model reflects some of the histological and immunological features of multiple sclerosis (MS) in humans. Mice genetically engineered to label Ccr2 with red fluorescent protein (rfp) and Cx3cr1 with green fluorescent protein (gfp) (“red-green mice” as described in Saederup et al., 2010 and Mizutani et al., 2012) were employed and microglia (positive for gfp) were separated from infiltrating monocytes using flow cytometry. Overall design: Microglia cells were purified and pooled from the brains of 30 mice per condition for both the naïve (before EAE induction) and peak (peak of EAE induction) timepoints of the EAE mouse model. DNA and RNA were extracted, and RNA-seq and MiGS were performed for each timepoint. MiGS was performed for two biological replicates. Due to the difficulty of obtaining the materials, the RNA-seq was not replicated.