library_construction_protocol: scATACseq Gastrula samples: Nuclei from 300 pooled gastrula were isolated in 1x OmniATAC lysis buffer supplemented with Pitstop2 (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 1%BSA, 0.1% NP-40, 0.1% Tween-20, 0.01% digitonin, 70uM Pitstop2), 3min while gently dounce homogenizing in cold conditions. Nuclei were then wash it in ATACwash buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 2%BSA, 0.1% Tween-20), twice in cold PBS-1%BSA, and finally gently resuspended in 1x Diluted buffer from 10x genomics (sample 1_Gastrula_Fresh). When nuclei from gastrula samples were fixed, nuclei were isolated in 1x OmniATAC lysis buffer without digitonin and mildly fixed in 0.1% methanol-free formaldehyde (PFA) prior to purification by FACS (FACS Influx, 100um nozzle, 12psi). Then, sorted nuclei were  permeabilized in 0.1x OmniATAC lysis buffer with Pitstop2 for 2min on ice while gently pipetting (sample 2_Gastrula_Fix). After washing, nuclei were processed as described above for fresh nuclei. scATACseq Adult samples: 2-4 adult polyps were gently dounce homogenized in ice-cold TST lysis buffer (10mM Tris-HCl pH 7.5, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 0.03% Tween-20, 1x cOmplete protease inhibitor) until a homogenous nuclei suspension was achieved for a maximum of 12min. The resulting nuclei suspension was wash it in ST-2%BSA buffer (without tween-20) and mildly fixed in 0.1% PFA prior to FACSorting (FACS Influx, 100um nozzle, 12psi). FACS-purified nuclei were then permeabilized in 0.1x OmniATAC lysis buffer with Pitstop2 for 2min on ice while gently pipetting, wash it and resuspended in 1x diluted buffer from 10x genomics (samples 3-15_Adult_Fix; samples 17-19_Adult_Fix). When nuclei from adult samples were processed fresh (without PFA fixation), NP-40 was added to TST lysis buffer (0.01% NP-40 final concentration) after dounce homogenization and further dissociated 5min by pipetting. In this case, nuclei were purified from debris using an OptiPrep® continuous density gradient. Fresh purified nuclei were permeabilized in ice-cold 1x OmniATAC lysis buffer with Pitstop2 for 4min while gently pipetting, wash it and resuspended in 1x diluted buffer from 10x genomics (sample 16_Adult_Fresh). scATACseq Elav+ neurons: One month old NvElav1::mOrange positive polyps were dissociated at 25°C in calcium- and magnesium-free NM (CMF/NM) containing 5mM EDTA and 0.25% α-chymotrypsin . Single cell suspensions were then stained with Hoechst 33342 (1µg/ml) and TO-PRO-3 (50nM) to remove debris and non-viable cells by FACS (FACS Aria II, 100um nozzle). Nuclei from sorted mOrange-positive cells were isolated, mildly fixed in 0.1%PFA and permeabilized (samples 20-21 Elav_fix), or directly permeabilized in OmniATAC lysis buffer with Pitstop2 (samples 22-24 Elav_fresh). Finally, permeabilized nuclei were washed in ATAC wash buffer, resuspended in 1x Diluted buffer and encapsulated using 10x Chromium platform. scMultiome (ATAC+GEX): 2 adult wild-type polyps were dissociated and stained for FACSorting as described above for NvElav1::mOrange samples. Nuclei from single viable cells were isolated and permeabilized for 3min in ice-cold 0.1x OmniATAC lysis buffer supplemented with Pitstop (70mM), RNAse inhibitor (1U/ul) and DTT (1mM). Nuclei were then washed in ATAC wash buffer, resuspended in 1x Diluted Nuclei Buffer with RNAse inhibitor (1U/ul) and DTT (1mM), and encapsulated using 10x Chromium platform. scRNAseq 5' GEX: A single cell suspension from 2-3 month old polyps were obtained after ACMEsorbitol (0.4M) fixation and dissociation as previously described (Garcia-Castro, H., et al., 2021; Najle,S.R and Grau-Bove,X., et al, 2023). scATAC-seq libraries from whole-gastrula stage were prepared using the Chromium scATAC v2 (Next GEM) kit from 10x Genomics, following the manufacturer's instructions scATAC-seq libraries from whole-adult and elav+ cells were prepared using the Chromium scATAC v1.1 (Next GEM) kit from 10x Genomics, following the manufacturer's instructions scMultiome, libraries (ATAC+GEX) from whole-adult cells were prepared using the Chromium single cell multiome kit (Next GEM) from 10x Genomics, following the manufacturer's instructions. 8 PCR cycles were performed in Step 5.1 for ATAC library construction. 9 PCR cycles of cDNA amplification were performed in Step 6.1 for GEX library construction scRNAseq 5' GEX library from whole-adult cells was prepared using the Chromium Next GEM 5' GEX scRNAseq v2 kit from 10x Genomics, following the manufacturer's instructions and with 14 PCR cycles of cDNA amplification