design: For the CUT&RUN assay, uninjured mature limb tissue (DMSO treated), 7-day-old A-lateral wound tissue (DMSO treated), and ND-A blastema tissue for each treatment condition (DMSO, LDN193189, SU5402, and LDN193189+SU5402 treated) was collected in quadruplicate from 2-3 limbs per sample. Wound epithelia were removed from the blastema mesenchyme, cells were counted on a hemocytometer, and 80,000-100,000 connective tissue cells were collected per sample. Three of the collected samples per condition were designated biological replicates, and the fourth was set aside as an input sample. Samples were processed following the manufacturers protocol for the CUT&RUN Assay Kit (86652, Cell Signaling), and 5 L of the anti-H3K27me3 antibody (A4039050, Epigentek) was used for each biological sample in incubation. Input samples were fragmented using Cell Signalings MNase Input sample fragmentation protocol. Sample and input DNA was then purified using Cell Signaling DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) (Cell Signaling) and eluted in 50 L of DNA Elution Buffer. DNA libraries were prepared and barcoded for multiplexing using the Cell Signaling DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) (56795, Cell Signaling) and Multiplex Oligos for Illumina (Dual Index Primers) (ChIP-seq, CUT&RUN) (47538, Cell Signaling). Samples were then validated using the Agilent 2100 Bioanalyzer system. Sequencing was conducted using the HiSeq 2500 System, rapid Run 1 x 51, per 2 lane FC.