The immediate objective of our research is to generate a method that will enable researchers to multiplex Chromatin ImmunoPreciptation-Sequencing (ChIP-Seq) analysis in a single Next generation DNA sequencing run. In the to-be-developed method: i) a set of antibodies directed against specific DNA-binding proteins are uniquely bar-coded with a DNA 'ZipCode;'ii) covalent cross-links between DNA-binding proteins and chromosomal DNA are formed by treating cells with formaldehyde;iii) the set of DNA-barcoded antibodies specific to the proteins of interest are used to selectively coimmunoprecipitate the protein-bound DNA fragments that were covalently cross-linked;iv) excess antibodies and chromosomal DNAs are removed by washing;v) the enriched protein-bound chromosomal DNA is ligated to the antibody-attached ZipCode DNA using T4 DNA ligase, and;vi) the immunoprecipitated protein-DNA links are reversed and the recovered DNA is assayed using Next-generation sequencers to determine both the chromosomal DNA sequence bound by the protein and the antibody-identifying DNA ZipCode.
Researchers will benefit from an increased understanding of the function of human proteins. Methods are needed that can facilitate this understanding. The immediate objective of our research is to generate a method that will enable researchers to multiplex the method known as Chromatin ImmunoPreciptation-Sequencing (ChIP-Seq) analysis, in a single DNA sequencing run. We anticipate using our high-throughput antibody-discovery pipeline for producing recombinant antibodies as a means to generate the reagents needed to make this multiplexed method possible.
