Dissection of forelimb buds from wild type embryos and Hox13-/- embryos were performed at E11.5. Forelimbs were dissected in cold 1XPBS, after centrifugation at 300 rcf for 5 minutes at 4°C the forelimbs were incubated in dissociation buffer (450 μl of 0.25% Trypsine/EDTA (GIBCO), 50 μl 10% BSA, 1 μl DNAseI (NEB)) for 10 minutes at 37°C. After incubation limb cells were gently mixed by pipetting up and down 10-15 times until they were dissociated, then 10% final FBS was added. Dissociated cells were filtered using a cell strainer (40 μm Nylon, BD Falcon) and after centrifugation at 300 rcf for 5 minutes at 4°C the resulting pellet of cells was resuspended in 1XPBS, 0.1%BSA. Dissociated cells were counted and assessed for cell viability using 0.4% Trypan blue. After centrifugation at 300 rcf for 5 minutes at 4°C, the cell pellet was resuspended in 100 μl of ice-cold lysis buffer (10mM Tris-HCl, pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% Digitonin, 1% BSA) and incubated for 5 minutes on ice. Then 1 ml of wash buffer (10mM Tris-HCl, pH7.4, 10 mM NaCl, 3mM MgCl2, 0.1% Tween-20, 1% BSA) was added to the cells. After centrifugation at 300 rcf for 5 minutes at 4°C, the pellet of nuclei was re-suspended in diluted nuclei buffer in order to have 3500 nuclei/μl and processed using Chromium Single Cell ATAC Reagent Kits (10x Genomics, Pleasanton, CA) following the manufacturer recommendation. nuclei were incubated in a Transposition Mix that includes a Transposase that preferentially fragments the DNA in open regions of the chromatin and simultaneously, adapter sequences were added to the ends of the DNA fragments. GEMs were then generated, using partitioning oil, in order to contain a single nucleus and a Master Mix to produce 10x barcoded single-stranded DNA after thermal cycling. The Chromium Single Cell ATAC Library was amplified and double sided size selected. Samples were controlled at multiple steps during the procedure by running on BioAnalyzer. Libraries were sequenced on Hiseq 4000 with 100 bp paired- end reads scATACseq using 10x Chromium Single Cell ATAC Reagent Kit