Microglia modulate TNFα‐mediated synaptic plasticity

Transcriptome analysis Tissue cultures (n = 3) were transferred as one sample into 250 μL RNAlater (Thermo Fisher Scientific, Cat# AM7020) and stored at −20°C. RNA was isolated after homogenization with TRIzol (Thermo Fisher Scientific, Cat# 15596018) using the Direct-zol RNA Microprep-Kit (Zymo Research, Freiburg im Breisgau, Germany, Cat# R2061) according to the manufacturer's instructions. RNA was eluted in 50 μL water and precipitated with 0.75 M ammonium-acetate and 10 μg glycogen (Thermo Fisher Scientific, Cat# R0551) by adding 125 μL ethanol (100%). Samples were incubated at −80°C overnight and consecutively centrifuged for 30 min at 4°C. Pellets were washed with 70% ethanol, centrifuged again and dried. Finally, pellets were dissolved in water for further processing. RNA concentration and integrity were consecutively analyzed by capillary electrophoresis using a Fragment Analyzer (Advanced Analytical Technologies, Inc., Ankeny, IA, USA) and the Agilent RNA 6000 Pico Kit (Agilent, Cat# 5067-1513). RNA samples with RNA integrity numbers (RIN) > 8.0 were further processed with the Affymetrix WT Plus kit and hybridized to Clariom S mouse arrays (Thermo Fisher Scientific, Cat# 902931) as described by the manufacturer. Briefly, labeled fragments were hybridized to arrays for 16 h at 45°C, 60 rpm in a GeneChip™ Hybridization Oven (Thermo Fisher Scientific). After washing and staining, the arrays were scanned with the Affymetrix GeneChip Scanner 3000 7G (Thermo Fisher Scientific). CEL files were produced from the raw data with Affymetrix GeneChip Command Console Software Version 4.1.2 (Thermo Fisher Scientific). CEL files were processed with the Oligo R package and RNA and intensity were normalized using Robust Multichip Average method. A linear-model-based analysis, limma R package (Ritchie et al., 2015), was used to identify differentially regulated genes. An adjusted p-value (Benjamini & Hochberg) below 0.05 was considered as significant. Gene-set enrichment analysis was done using GSEA 4.1.0 (Mootha et al., 2003; Subramanian et al., 2005), Cytoscape (Shannon et al., 2003) and custom R-scripts to produce heatmaps.