library_construction_protocol: For each mouse sample, five limbs from ALI or 96-well explant cultures were collected in Protein LoBind tubes (Eppendorf, 0030 108 442). Limbs were washed with 1 ml PBS 1x (Gibco, 10010-015) and incubated in 200 µl 0.25% trypsin-EDTA (Gibco, 25200072) at 37 ºC for 5-6 minutes. Following mechanical dissociation by a P200 pipette, trypsin was deactivated by the addition of 400 µl of FBS-containing media. Each sample was topped up with an additional 600 µl of PBS 1x and centrifuged at 400 rcf for 5 minutes at 4 ºC.  For each xenopus sample, eight limbs from ALI or 96-well explant cultures were collected in Protein LoBind tubes. Limbs were washed with 0.7X PBS and treated with 0.35 mg/mL Liberase solution (Sigma-Aldrich, 05401127001) diluted in 0.7X PBS for 30 minutes at room temperature while gently rotating. Cells were mechanically dissociated by pipetting 10–15 times until visibly dislodged. Next, 1 mL of L15 medium (supplemented with 1% antibiotic-antimycotic, 0.8 mM L706 glutamine, and 20% FBS) was added to the mixture. The cells were then centrifuged at 600 rcf for 5 minutes at room temperature and washed with 1 mL of 0.7X PBS containing 0.04% BSA. CUT&Tag libraries were prepared as previously reported (Zenk et al., 2023). The centrifuged cells were resuspended in 1 ml of CUT&Tag wash buffer (20 mM HEPES [pH 7.5] (Jena Bioscience, BU-106-75), 150 mM NaCl (Sigma-Aldrich, 71386), 0.5 mM spermidine (Merck, S2626), 5 mM sodium butyrate (Sigma-Aldrich, 303410), and 1x Protease Inhibitor Cocktail (Roche, 11873580001) in UltraPure distilled water (Invitrogen, 10977-035)). Then, a volume of Concanavalin A beads (Polysciences, 86057-3) corresponding to 15 µl per histone mark target was prepared by removing the supernatant on a magnetic rack and washing in 1 ml binding buffer (20 mM HEPES [pH 7.5], 10 mM KCl (Sigma-Aldrich, 60142), 1 mM CaCl2 (Sigma-Aldrich, 21115), and 1 mM MnCl2 (Sigma-Aldrich, M1028) in UltraPure distilled water). The supernatant was removed again, and the beads were resuspended in the original volume (15 µl per sample) of binding buffer.  Beads were added to the cell suspension for binding. Using all cells from the dissociated limbs, this corresponded to 60k-600k live cells per histone mark. After 15 minutes of incubation at room temperature on a rotating wheel, tubes were placed in a magnetic rack, the supernatant was discarded, and cells were permeabilized in wash buffer supplemented with 0.01% Digitonin (Invitrogen, BN2006) until nuclei were visible around the beads via Trypan Blue (Invitrogen, T10282) staining.  The supernatant was discarded again on the magnetic rack, and samples were resuspended in 150 µl wash buffer per histone mark and distributed in separate tubes for antibody binding. Primary antibodies were added as per the manufacturer's suggested concentration for CUT&Tag (H3K27me3 (Diagenode, C15410195): 1 µg in 150 µl; H3K4me3 (Diagenode, C1541003): 0.5 µg in 150 µl; H3: dilute at 1:150 (Activemotif, 39763) along with 2 mM EDTA (Invitrogen, 15575-038). Samples were incubated overnight at 4 ºC on a nutator.  The next day, the primary antibody solution was removed on a magnetic rack, and the beads were washed twice with 400 µl of CUT&Tag wash buffer, vortexing briefly each time. Beads were resuspended in 150 µl of CUT&Tag wash buffer with 1.5 µl of secondary antibody (Abcam, 46540; Antibodies Online, ABIN101961), and incubated for 1 hour at 4 ºC on a nutator.  Next, the secondary antibody solution was removed on a magnetic rack, and the beads were washed twice with 400 µl of CUT&Tag wash buffer, vortexing briefly each time. Beads were resuspended in 150 µl of high salt wash buffer (20 mM HEPES [pH 7.5], 300 mM NaCl, 0.5 mM spermidine, 5 mM sodium butyrate, and 1x Protease Inhibitor Cocktail in UltraPure distilled water) with 2 µl of homemade Protein A-Tn5, and incubated for 1 hour at room temperature in a thermomixer at 300 rpm.  The Protein A-Tn5 solution was removed on a magnetic rack, and the beads were washed twice with 400 µl of high salt wash buffer. Beads were resuspended in 100 µl CUT&Tag wash buffer supplemented with 10 mM MgCl2 (Invitrogen, AM9530G) for chromatin digestion for 1 hour at 37 ºC in a thermomixer at 300 rpm. The reaction was stopped by adding 4.5 µl of 0.5 M EDTA, 2.76 µl of 20% SDS (Sigma-Aldrich, 05030), and 1 µl of 20 mg/ml Proteinase K (Invitrogen, AM2546), and incubating in a thermomixer at 300 rpm for 30 minutes at 55 ºC, followed by 20 minutes at 70 ºC.  Each sample was then purified using the ChIP DNA Clean & Concentrator kit (Zymo, D5205) as per the manufacturer's instructions for a 100 µl sample. At the final step, samples were eluted in 25 µl of elution buffer (Qiagen, 19086) containing 2 pg of lambda-DNA (NEB, N3011S; tagmented with Tn5 and cleaned up on columns before use) as a spike-in.  For library preparation, 21 µl of each sample was transferred to PCR tube strips, and 25 µl of Nebnext® High-Fidelity 2X PCR Master Mix (NEB, M0541S) was added, along with 2 µl of each i7 and i5 index primer. The following thermocycler program was used: 1x 5 min at 58 °C (gap filling); 1x 5 min at 72 °C (gap filling), 1x 30 s at 98 °C (denaturation), 14x 10 s at 98 °C (denaturation) plus 30 s at 63 °C (annealing/extension), 1x 1 min at 72 °C (extension), hold 4 °C.  A final clean-up was performed by mixing in 45-55 µl of AMPure XP beads (Beckman Coulter, A63881) and incubating at room temperature for 15 minutes. The supernatant was removed on a magnetic rack, and the beads were washed twice in 400 µl 80% EtOH. Finally, beads were resuspended in 33 µl 0.1x TE buffer and incubated for 5 minutes at room temperature. On a magnetic rack, 28 µl of the supernatant was transferred to a new tube. Quality control was performed using the Qubit dsDNA HS Assay kit (Invitrogen, Q32854) and the Agilent High Sensitivity D5000 ScreenTape system (ScreenTape: Agilent, 5067-5592; Reagents: Agilent, 5067-5593).  An equimolar pool (5-15 nM) of samples was submitted for sequencing.