SRX5392461 GSM3610370 SRP186305 SRS4380284 GSM3610370 RNA-Seq TRANSCRIPTOMIC cDNA Nkx2.5:ZsYellow+ cells from control and Pbx4-depleted embryos were isolated via FACS at 28 hpf. Dissociation of embryos was performed as described by (Stachura and Traver, 2016) with the following modifications, approximately 180 embryos per condition were manually dechorionated and rinsed in ice cold Hank's Balance Salt Solution (HBSS) containing Ca2+ and Mg2+. Embryos were subsequently transferred to 1.5ml tubes in 500 µl HBSS and dissociation was initiated by adding 60 µl Liberase (Roche). Embryos were deyolked by pipetting up and down, first with a p1000 tip and subsequently with a p100 tip, as described by (Samsa et al., 2016). Following yolk removal, embryos were incubated at 32.5°C. To facilitate dissociation, embryos were gently homogenized using a handheld pestle homogenizer (5-10 seconds) and returned to 32.5°C. This process was repeated until complete dissociation was achieved which took approximately 15 minutes. Dissociation of embryos was monitored using a dissecting microscope. Following dissociation, cell suspensions were centrifuged for 5 minutes at 13,000 rpm at 4°C. The supernatant was discarded and the resulting pellets were resuspended in 500 µl of FACS medium consisting of 0.9X PBS and 2% fetal bovine serum (FBS). Samples were filtered twice through a 40 µm strainer and collected in a FACs tube. FACS sorting was performed by the CCHMC Research Flow Cytometry Core using a 70 µm nozzle and a gating strategy as described by (Samsa et al., 2016). The 10X Genomics Chromium Instrument and cDNA synthesis kit (10x Genomics) was used by the CCHMC Genomics core to generate a barcoded cDNA library for single cell RNA-sequencing from approximately 4,000 cells. cDNA library quality was determined using an Agilent Bioanalyzer. Using this library, we performed one full lane sequence on two paired-end 75bp Flow Cells using on an Illumina HiSeq2500. 10X Genomics Chromium v2 chemistry Illumina HiSeq 2500 gds 303610370 GSM3610370 GEO Accession GSM3610370 SRX5392462 GSM3610371 SRP186305 SRS4380285 GSM3610371 RNA-Seq TRANSCRIPTOMIC cDNA Nkx2.5:ZsYellow+ cells from control and Pbx4-depleted embryos were isolated via FACS at 28 hpf. Dissociation of embryos was performed as described by (Stachura and Traver, 2016) with the following modifications, approximately 180 embryos per condition were manually dechorionated and rinsed in ice cold Hank's Balance Salt Solution (HBSS) containing Ca2+ and Mg2+. Embryos were subsequently transferred to 1.5ml tubes in 500 µl HBSS and dissociation was initiated by adding 60 µl Liberase (Roche). Embryos were deyolked by pipetting up and down, first with a p1000 tip and subsequently with a p100 tip, as described by (Samsa et al., 2016). Following yolk removal, embryos were incubated at 32.5°C. To facilitate dissociation, embryos were gently homogenized using a handheld pestle homogenizer (5-10 seconds) and returned to 32.5°C. This process was repeated until complete dissociation was achieved which took approximately 15 minutes. Dissociation of embryos was monitored using a dissecting microscope. Following dissociation, cell suspensions were centrifuged for 5 minutes at 13,000 rpm at 4°C. The supernatant was discarded and the resulting pellets were resuspended in 500 µl of FACS medium consisting of 0.9X PBS and 2% fetal bovine serum (FBS). Samples were filtered twice through a 40 µm strainer and collected in a FACs tube. FACS sorting was performed by the CCHMC Research Flow Cytometry Core using a 70 µm nozzle and a gating strategy as described by (Samsa et al., 2016). The 10X Genomics Chromium Instrument and cDNA synthesis kit (10x Genomics) was used by the CCHMC Genomics core to generate a barcoded cDNA library for single cell RNA-sequencing from approximately 4,000 cells. cDNA library quality was determined using an Agilent Bioanalyzer. Using this library, we performed one full lane sequence on two paired-end 75bp Flow Cells using on an Illumina HiSeq2500. 10X Genomics Chromium v2 chemistry Illumina HiSeq 2500 gds 303610371 GSM3610371 GEO Accession GSM3610371