Extended Data Fig. 2: Stable DARs are predominantly enriched for enhancers marks.

a, Workflow for the flow cytometry dissection of different neuronal population from the hippocampus, during memory formation and retrieval. Representative FACS plots showing expression of all population (left panel). Further selection was made on single nuclei and NeuN + /DAPI + population (middle panel). Last, selection was made on the gated sub-population; GFP + (adjusted to ~2.5% from all cells), ARC + /GFP + (~0.15% from all cells) and nuclei were sorted to 1.5 ml Eppendorf tubes coated with 200ul of 1% PBS. b, Venn Diagram (left) and table (right), which illustrate the overlap between the DARs identified in the different pairwise comparisons during memory formation and recall. c, Resampling-based statistical analysis was performed to determine if the enrichments of chromatin states over ChromHMM emissions (observed) are statistically significant. Expected enrichment was calculated by performing 10,000 randomized sets of overlaps (permutations) between ‘all accessible sites’ and ‘all histone modifications sites (that is all emissions)’ and presented as histogram in the figure. Sample size of each randomized set was determined by the size of DARs from each state. The mean and standard deviation of the sample was calculated (Supplementary Table 3). The number of observed overlaps between DARs and each emission was calculated and presented as lines. z-score was calculated as fallows; Z = (observed values (X)—mean of the sample (μ))/(standard deviation of the sample (σ)). z-score Basal vs. Early; S.E 10.5, W.E 6.9. Stable; S.E 16.9, W.E 12.7, all p < 0.0001, z-score Early vs. Late; S.P 91.7, W.P 38.7. Late vs. Reactivated; S.P 26.8, W.P 28.9, all p < 0.0001. p-values (Two-Sided) were calculated from z-table. Full analysis is reported in Supplementary Table 3. d, Pie chart shows percentage of different enhancer states, for all stable regions. Overlap of each individual stable region was performed with previously published H3K4me1 and H3K27ac ChIP-seq data, obtained 1 h after FS 29²¹. Enhancers states were classified as ‘primed’—overlap with regions marked with only H3K4me1, ‘active’—with H3K4me1/H3K27ac or ‘latent’—no overlap. e, Motifs identified from nucleosome free regions (NFR) on the ATAC-seq tracks from each state (Basal, Activated-early, Activated-late and Reactivated). Peaks were divided into positions that annotated to promoters (5 kb from TSS) and enhancers (>5 kb from TSS). Circle size indicate percentage of enrichment (1–50%). Color indicates –log(P-value).