RNA seq data analysis

Total RNA extraction from sorted human microglia followed a methodology similar to the one detailed earlier. The study encompassed six distinct experimental groups. For each treatment and vehicle group, sorted human microglia were gathered from four animals. Consequently, the RNAseq analysis involved a total of 24 animals distributed across six experimental categories: 1-week treatment of APOE ASO-1, 4-week treatment of APOE ASO-1, 1-week treatment of TREM2 ASO-171, 4-week treatment of TREM2 ASO-171, and the respective vehicle groups for both 1 week and 4 weeks. RNA samples were then sent to BGI (bgi.com) for library preparation and sequencing. Samples were sequenced on their DNBSEQ-G400 platform. Sequencing data were mapped using STAR version = 2.7.10 against the joined reference library by combining the Mus musculus reference genome (mm10/GRCm38) and the human genome (GRCh38). The full count matrix was produced by FeatureCount v1.6.3 from the Subread package [68], using reads mapped to the human genome. We conducted differential expression analysis comparing the treatment and vehicle groups using the DEseq package [69], which streamlines standard differential expression analysis procedures in a single function DEseq. In this analysis, a design matrix was constructed for each treatment group, with the corresponding duration-matched vehicle group as the reference group. We then employed the Wald test to ascertain the log2fold changes and associated p-values for the comparison. To control for multiple testing, we applied the false discovery rate (FDR) adjustment using DEseq2's default Benjamini–Hochberg procedure.