GSM6559647: FL_4C_Sox9_bait_rep2; Mus musculus; OTHER
4C-seq libraries were generated from microdissected E14.5 Meckel's cartilage and forelimb cartilage as described previously(PMID: 31351925). Cells were crosslinked with 1% formaldehyde for 10 mins at room temperature, then treated with 0.2M glycine. The crosslinked cells were pelleted, washed with HBSS, and incubated with lysis buffer (10mM Tris-HCl pH 8.0, 10mM NaCl, 0.2% Igepal CA-630 with cOmplete Protease Inhibitor Cocktail). Lysates were pelleted at 2500x g for 5min, washed with 1x NEB CutSmart Bffer, and chromatin was then opened up at the presence of 0.5% SDS at 65°C for 5mins and quenched with Triton X-100 at the final concentration of 1%. Samples were then digested overnight with Nlalll at 37°C. First round of ligation was carried in a total volume of 1.2ml with 50U T4 DNA ligase at 16°C for 4hr. The ligated products were then treated with 20 mg/ml Proteinase K overnight to reverse crosslinking. To generate 3C libraries, DNA purification was performed by phenol-chloroform isoamyl alcohol extraction and ethanol precipitation. 3C libraries were then treated with DpnII overnight at 37°C for the second round of restriction enzyme digestion, followed by overnight ligation at 16°C. The DNA was purified by phenol-chloroform isoamyl alcohol extraction and ethanol precipitation, and clean-up of 4C-seq libraries was then performed with PCR purification kit (Qiagen). Quality controls of the first and second rounds restriction enzyme digestion and ligation were assessed by gel electrophoresis.  Amplification of 4C-seq libraries was carried out with two rounds of PCR with the specificity of primers checked with test PCR. The first PCR step is to amplify the fragments ligated to Sox9 promoter viewpoint using the Expand Long Range PCR System (Roche). A total of four PCR reactions with 200 ng 4C-seq template per reaction was performed. 50μl aliquot of all pooled PCR reactions was purified with AMPure beads at 0.8x volume. The purified PCR products of the first round were amplified by universal primers containing the Illumina adapters required for flow cell binding of the amplicons, and then sequenced on the NovaSeq platform after purification with PCR purification kit (Qiagen).