The study describes the methods used to isolate RNA from hippocampi, striata, and cortex of Aldh1l1-eGFP-L10a mice, followed by RNA sequencing and analysis. RNA was extracted from 20% of cleared lysate as input, while the remaining lysate was incubated with anti-GFP magnetic beads to isolate TRAP RNA. RNA was purified using the RNeasy Plus kit and assessed for quality. RNAseq libraries were constructed from total RNA and TRAP-isolated samples using the Ovation RNAseq system and Next Ultra RNAseq library prep kit. Libraries were sequenced on an Illumina NextSeq sequencer to obtain 75 bp paired-end reads. RNAseq analysis was performed using the Tuxedo pipeline on Galaxy. Reads were aligned with Bowtie2 and splice junctions identified with TopHat. Expression levels were estimated using Cufflinks. Gene expression data (FPKM) were downloaded and merged into datasets S1–S4. IPA was used to analyze pathway enrichment based on FPKM, log2(Fold Change), P values, and FDRs from Cufflink and edgeR analyses. Microfluidics-based qPCR was used to quantify gene expression. Total RNA was extracted, cDNA synthesized, and primers designed to span exon-exon junctions to avoid amplification of genomic DNA. PCR specificity was tested with mouse whole-brain cDNA. Preamplification was performed, followed by qPCR on a 96.96 Dynamic Array chip. Data were collected and analyzed using Fluidigm software. Cell-type-specific transcripts were also detected for microglia, oligodendrocyte lineage cells, and neurons. Figures S1–S6 show the characterization of aging-induced transcriptional changes, reactive gene changes, and the LPS response in astrocytes. Datasets S1–S4 provide differentially expressed genes in aged hippocampal, striatal, and cortical astrocytes, as well as RNAseq data for all astrocyte samples across the mouse lifespan.The study describes the methods used to isolate RNA from hippocampi, striata, and cortex of Aldh1l1-eGFP-L10a mice, followed by RNA sequencing and analysis. RNA was extracted from 20% of cleared lysate as input, while the remaining lysate was incubated with anti-GFP magnetic beads to isolate TRAP RNA. RNA was purified using the RNeasy Plus kit and assessed for quality. RNAseq libraries were constructed from total RNA and TRAP-isolated samples using the Ovation RNAseq system and Next Ultra RNAseq library prep kit. Libraries were sequenced on an Illumina NextSeq sequencer to obtain 75 bp paired-end reads. RNAseq analysis was performed using the Tuxedo pipeline on Galaxy. Reads were aligned with Bowtie2 and splice junctions identified with TopHat. Expression levels were estimated using Cufflinks. Gene expression data (FPKM) were downloaded and merged into datasets S1–S4. IPA was used to analyze pathway enrichment based on FPKM, log2(Fold Change), P values, and FDRs from Cufflink and edgeR analyses. Microfluidics-based qPCR was used to quantify gene expression. Total RNA was extracted, cDNA synthesized, and primers designed to span exon-exon junctions to avoid amplification of genomic DNA. PCR specificity was tested with mouse whole-brain cDNA. Preamplification was performed, followed by qPCR on a 96.96 Dynamic Array chip. Data were collected and analyzed using Fluidigm software. Cell-type-specific transcripts were also detected for microglia, oligodendrocyte lineage cells, and neurons. Figures S1–S6 show the characterization of aging-induced transcriptional changes, reactive gene changes, and the LPS response in astrocytes. Datasets S1–S4 provide differentially expressed genes in aged hippocampal, striatal, and cortical astrocytes, as well as RNAseq data for all astrocyte samples across the mouse lifespan.