Agilent 2200 tapestation assay

Percoll-enriched microglia were sorted using a Becton-Dickinson FACSAria III cell sorter at the OSUCCC Analytical Cytometry facility. Microglia were identified by CD11b⁺/CD45^low expression. Cells were pelleted and lysed in Arcturus PicoPure Extraction Buffer immediately after sorting. The Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) was used to purify and concentrate total RNA from sorted microglia. During RNA isolation, samples were treated with on-column DNase digestion for 15 min at 23 °C to eliminate contaminating genomic DNA. A 1-mm coronal brain section was also collected from each brain and snap frozen in liquid nitrogen. RNA was isolated using the Tri-Reagent protocol (Sigma-Aldrich). RNA quality and integrity was determined using the Agilent 2200 TapeStation assay (Agilent Technologies). RNA-Seq was performed on sorted microglia and brain section (Bregma - 1.5 mm) RNA at the Hussman Institute for Human Genomics Sequencing Core Facility (University of Miami, Miami, FL). Briefly, RNA-Seq libraries were prepared using the Ovation SoLo RNA-Seq System with AnyDeplete rRNA to remove rRNA and other abundant transcripts according to the manufacturer’s recommendation (Nugen). RNA-Seq libraries were run on an Illumina NextSeq 500 sequencing instrument according to the protocols described by the manufacturer.

Mice were sacrificed by CO₂ asphyxiation and the hippocampus was dissected and snap frozen in liquid nitrogen (− 196 °C). Hippocampal RNA was isolated using the Tri-Reagent protocol (Sigma-Aldrich). RNA quality and integrity was determined using the Agilent 2200 TapeStation assay (Agilent Technologies). nCounter analysis (NanoString Technologies) was performed by the OSU Comprehensive Cancer Center (OSUCCC) Genomics Shared Resource facility (The Ohio State University, Columbus, OH) using the Mouse Inflammation v2 Panel for 248 inflammation-related mouse genes, 20 custom genes, and 6 internal reference controls. Copy numbers were normalized using DESeq2 Bioconductor package in R [41 (link)].