The AVCs and limb buds were isolated from E10.5/12.5 embryos in PBS. AVCs were manually dissected out from the heart. Tissues were homogenized in 1% formaldehyde and incubated at room temperature (RT) for 10 min. Samples were incubated with 0.125 M glycine at RT for 5 min. Following pelleting and washing, samples were resuspended in 5 cell pellet volumes of ChIP cell lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) with a protease inhibitor cocktail (PIC, Roche). Cells were re-homogenized and put on ice for 15 min. Cells were pelleted again and resuspended in 3 volumes of ChIP nuclear lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris-H Cl, pH 8.1) containing a protease inhibitor cocktail (Roche). Following a 30-60 min incubation on ice, samples were sonicated (Sonicator 3000, Misonix) in an ice water bath for 20 cycles of 30 s on, 40 s off. After a 10 min centrifugation (4˚C, 13.2krpm) to remove cellular debris, sonicated chromatin was precleared with 20 μl Protein A/G beads (ThermoFisher Scientific, 53135), 10 μl 10x PIC and ChIP dilution buffer (with a volume equal to 4x that of the chromatin volume used, 0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 16.7 mM Tris–Cl, pH 8.1) for 1 hr, 4˚C with rotation. After 2 min 4000 rpm centrifugation, diluted chromatin removed from beads and transferred to new tubes and antibodies were added as follows: 3 μg of rabbit polyclonal anti-mouse SOX9 antibody (Millipore AB5535) or for negative controls 3 μg of normal rabbit IgG antibody (Santa Cruz, sc-2027). Samples were incubated overnight while rotating at 4˚C. Simultaneously, 20 μl of fresh Protein A/G beads (ThermoFisher Scientific) were blocked with 1 mg/ml BSA and 0.1 mg/ml herring sperm DNA in ChIP dilution buffer overnight at 4˚C. The following day, samples were incubated with blocked Protein A/G beads for four hours rotating at 4˚C. Beads were washed with several buffers as follows: low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt buffer (low salt buffer with 500 mM NaCl), lithium chloride buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and two final washes with TE buffer. 125 μl elution buffer (1% SDS, 0.1 M NaHCO3) was added to remove the bound chromatin-antibody complexes from the beads and samples were rotated at RT for 15 min and after centrifugation the eluted solution was removed from the beads. This was repeated. To reverse the crosslinking and break the complex bonds, 10 μl 5M NaCl, 10 μl 1M Tris-HCl (pH 6.5), 5 μl 0.5M EDTA, 1 μl Proteinase K (20mg/ml), and 2.5 μl RNAseA (10mg/ml) was added to each of the samples and incubated at 65˚C overnight. Purification of DNA was performed with two rounds of phenol-chloroform extraction and ethanol precipitation. Samples were spun down and pellets were resuspended in 50 μl dH2O. A sample of unbound sonicated DNA served as input for sequencing. ChIP DNA was sent to the Genome Sciences Centre (Vancouver, BC) for library generation. DNA was purified using 8-12% PAGE to isolate 100-300bp fragments for short read (50bp) sequencing on an Illumina Genome Analyzer (GA2).