Tbx3Venus/+ hearts were dissected from embryonic day (E) 12.5-E14.5 embryos and AV conduction system tissues were isolated by microdissection. Tissue samples were dissociated into a single cell suspension using 0.05% Trypsin/EDTA (ThermoFisher Scientific, 25300-054) for 15 minutes at 37°C. Dissociated cells were resuspended in DMEM (ThermoFisher Scientific, 31966-021) supplemented with 10% FBS (ThermoFisher Scientific, 10270-106) and fluorescence activated cell sorting (FACS) performed selecting for cells with Venus fluorescence. Approximately 75.000 Venus+ cells were collected and used as input for assay for transposase-accessible chromatin (ATAC)-sequencing. Venus+ cells were washed in PBS and lysed in cold lysis buffer (10mM Tris.Cl, pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% (v/v) Igepal CA-630). Transposition reaction on the nucleus extract was performed as described using the Nextera DNA Library Prep kit (Illumina, FC-121-1030) for 30 minutes, followed by cleanup using the MinElute PCR Purification Kit (Qiagen, 28004). Transposed DNA fragments were amplified using NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs, M0541) with custom barcoded PCR primers (sequences as described in Buenrostro et al., 2013). The amplified library was purified using the MinElute PCR Purification Kit (Qiagen, 28004). Pooled libraries sequenced on a NextSeq 500 system (Illumina) with 76 bp single-end reads.