Figure EV4. Sorting neuronal and non‐neuronal nuclei for RNA‐Seq analysis.

Nuclei from the hippocampal CA region were subjected to FANS as depicted in Fig 2A.

1. Representative images showing nuclei that were sorted using the neuronal marker NeuN. Note that no NeuN positive nuclei are detected in the NeuN (−) fraction confirming the purity of the approach. Scale bar: 50 μm.
2. Gating strategy for NeuN (+) and NeuN (−) nuclei sorting.
3. RNA‐sequencing (n = 2/group) was performed from NeuN (+) and NeuN (−) nuclei and a differential expression analysis was performed. Heat map shows 836 genes specifically enriched in NeuN (+) nuclei when compared to NeuN (−) nuclei. The criteria to select those genes were: adjusted P‐value < 0.01, base mean > 150, fold change > 5.
4. GO‐term analysis showing that the top 10 enriched biological processes and molecular functions for the 836 genes enriched in NeuN (+) nuclei all represent specific neuronal processes.
5. Normalized expression values obtained from the RNA‐seq experiment showing the expression of selected genes known to be enriched in neurons.
6. Normalized expression values of genes that are known to be enriched in non‐neuronal cells including glia cells. Error bars indicate SEM.

Data information: Bar graphs indicate mean, Error bars indicate ± SEM. “n” indicates biological replicates.