Mogene 1.0 st array

The mouse microarray has been published in a previous study (Wang et al., 2015b (link)) and the publicly available dataset (GSE65067) was used. Briefly, microglia from female 8 month old wild-type (n = 3) and 5XFAD (n = 5) mice (The Jackson Laboratory) were FACS-sorted directly into RTL-plus lysis buffer. RNA extraction from microglia was performed using an RNeasy Plus Micro Kit (Qiagen, Cat. No. 74034). Microarray hybridization (Affymetrix MoGene 1.0 ST array) and data processing were performed at the Washington University Genome Center. Genes with P-values < 0.05 and log₂FC > 0.5 were considered differentially expressed.

Microglia were isolated from the indicated animals as previously described (Wang et al., 2015 (link)). CD45⁺, CD11b⁺, F4/80⁺ (Biolegend Cat. Number 103134, eBioscience Cat. Numbers 11-0112 and 17-4801) cells in the brain were fluorescence-activated cell-sorted (FACS) directly into RLT-plus lysis buffer for microarray or 2% FBS in PBS for TEM or immunoblot lysates. For microarray RNA extraction was performed using a RNeasy micro kit (QIAGEN). Microarray hybridization (Affymetrix MoGene 1.0 ST array) and data processing were performed at the Washington University Genome Center. For normalization, raw data was processed by Robust Multi-Array (RMA) method and genes were pre-filtered for expression value ≥ 120 expression units, a cut-off above which genes have a 95% chance of expression demonstrated in Immgen data set, which uses the same array platform(Wang et al., 2015 (link)). QIAGEN IPA analysis was preformed by comparing fold change and p-values for all genes 5XFAD and Trem2-deficient 5XFAD microglia. Heatmaps and hierarchical clustering were generated from preselected gene-lists using Morpheus. Microarray data has been deposited at GEO:GSE65067.