In vivo and in vitro Genome wide binding (ChIP-Seq) of Bapx1 and Sox9

Bapx1 was endogenously tagged with S-Peptide and dissected vertebral columns from these tagged embryos at E12.5 were subjected to immunoprecipitation by S-peptide antibody. Simultaneously V5 tagged Bapx1 was also over expressed in NIH_3T3 cells and immunoprecipitated with V5 antibody. Concurrently vertebral column fro E12.5 mouse embryos were subjected to immunoprecipitation with Sox9 antobody to compare binding sites of Sox9 and BApx1 in the vertebral column of developing mouse embryo Bapx1 was tagged with S-peptide tag by homologous recombination in mESC and stable mouse lines were generated. Similarly Bapx1 cDNA was cloned into the pCDNA 6 vector containing a n-terminal V5 tag and overexpressed in NIH_3T3 cells. Both these were used for immunoprecipitation with either V5 or S-pepetide to determine overall genome wide binding of Bapx1 and genome wide binding of Bapx1 in E12.5 vertebral column. For Sox9, endogenous Sox9 antibody was used to pull down Sox9 in veretebral column of E12.5 mouse embryo