## 2 Results  
### Total RNA Sequencing of Glia of Young and Aged Mice Identifies Differentially Regulated lncRNAs  
To identify deregulated lncRNAs in glia during aging, we isolated tissue from the hippocampal CA1 region of young (3 months) and old (16 months) mice. The hippocampus is a brain region crucial for memory function, and aging significantly affects this area. At 16 months, cognitive decline becomes observable in mice [3]. We processed the tissue to isolate nuclei via fluorescence-associated nuclei sorting (FANS), using neuronal nuclear protein (NeuN) to distinguish between neuronal (NeuN+) and non-neuronal nuclei (NeuN\(-\)) fractions. These two populations were then subjected to total RNA sequencing (Figure 1A).  
We aimed to identify lncRNAs that are enriched in the NeuN\(-\) fraction and deregulated during aging (Figure 1A). To achieve this, we first analyzed differentially regulated genes in the NeuN\(-\) populations of young and aged mice (Figure 1B,C). In comparison to young mice, 187 genes were upregulated, and 310 genes were downregulated in 16-month-old mice (\(\,\)Iog2 FC\(\,\mid\,>\,0.3\), FDR\(\,<\,0.05\), base mean \(>\,50\)) (Table S1). Filtering for lncRNAsfrom this list, we identified 17 upregulated and 7 downregulated lncRNAs (Figure 1D). Although our analysis focused on NeuN\(-\) nuclei, the 24 deregulated lncRNAs may also be expressed in NeuN+ nuclei. To determine if any of these lncRNAs were particularly enriched in the NeuN\(-\) population, we compared gene expression profiles of the NeuN+ and NeuN\(-\) fractions. Substantial differences were observed, with 51 lncRNAs highly enriched in NeuN\(-\) nuclei (Table S2).  
Next, we compared the list of lncRNAs deregulated during aging (\(u=24\)) with those enriched in the NeuN\(-\) fraction (n = 51). To facilitate the translation of our findings to human studies, we focused on lncRNAs with human homologs. Among the 24 deregulated lncRNAs in 16-month-old mice, seven were significantly enriched in the NeuN\(-\) fraction compared to NeuN+ nuclei (log2 FC \(>\) 4, padj \(<\) 0.05), with only three having human homologs. These included nuclear enriched abundant transcript 1 (_Neat1_) and  
Figure 1: Aging induces changes in glial lncRNA expression patterns. (**A**) Schematic illustration of the experimental approach of this study. (**B**) Heatmap showing gene expression changes in NeuN\(-\) nuclei isolated in 3- vs. 16-month-old mice. (**C**) Volcano plot showing the up- and downregulated coding transcripts when comparing Neu\(-\) nuclei from 3 vs. 16-month-old mice (log2 fold changes are depicted as 16/3 months). (**D**) Volcano plot showing expression changes in lncRNAs in Neu\(-\) nuclei (log2 fold changes are depicted as 16/3 months).  
3222401L13 Rik_, both upregulated in 16-month-old mice, and _C430049B03 Rik_, which was downregulated (Table 1). Since _Neat1_ has been extensively studied, particularly concerning its role in astrocytes [25], and _C430049B03 Rik_ has been analyzed as a host gene for miR-322, miR-503, and miR-531 in mouse embryonic fibroblasts [29], we chose to focus our study on _3222401L13 Rik_, a lncRNA that has not been previously characterized in any organ.