Transcriptome analysis of microglia and astrocytes in prion-infected mice by RNA-seq

Glial activation that is hallmark in prion disease precedes neurodegeneration and the onset of clinical disease. To estimate activation state of microglia and astrocytes in prion disease, cell-specific transcriptome analysis of microglia and astrocytes from brains in the Obihiro-, the Chandler- and mock-infected mice at 60, 90, 120 and 145 dpi (2 replicates each) were performed by RNA-sequencing. Cells were purified by magnetic-activated cell sorting. Total RNA was extracted from Astrocytes and microglia. Ribosomal RNA (rRNA) was depleted from of total RNA using Low Input RiboMinus Eukaryote System v2 (Thermo Fisher Scientific). For a removal of small RNA, rRNA-depleted RNA was subjected to LiCl precipitation. The whole transcriptome cDNA libraries were generated using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific). Samples were subjected to preparation of template by Ion OneTouch 2 Instrument and Ion One Touch ES module using Ion PI Template OT2 200 Kit v3 or Ion PI Hi-Q OT2 200 Kit (Thermo Fisher Scientific). Sequencing was carried out using with Ion PI Sequencing 200 Kit v3 and Ion PI Chip v2 or Ion PI Hi-Q Sequencing 200 Kit and Ion PI Chip v3 on Ion Proton System (Thermo Fisher Scientific).