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.
Public Health Relevance: 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.
Thesaurus Terms: Antibodies; Assay; Bar Codes; Base Pairing; Binding Proteins; Binding Sites; Bioassay; Biologic Assays; Biological Assay; Chip Assay; Cells; Chip (Chromatin Immunoprecipitation); Chemicals; Chromatin; Combining Site; Dna; Dna Sequence; Dna-Binding Proteins; Dna-Protein Interaction; Data; Deoxyribonucleic Acid; Development; Figs; Figs - Dietary; Formaldehyde; Formic Aldehyde; Gene Action Regulation; Gene Expression Regulation; Gene Regulation; Gene Regulation Process; Genetic Alteration; Genetic Change; Genetic Defect; Hand; Human; Human, General; Investigators; Length; Ligand Binding Protein; Link; Man (Taxonomy); Man, Modern; Maps; Methanal; Methods; Methods And Techniques; Methods, Other; Methyl Aldehyde; Modeling; Mutation; Nuclear Extract; Oxomethane; Process; Protein Binding; Proteins; Reactive Site; Reading; Reagent; Recombinant Antibody; Research; Research Personnel; Researchers; Running; System; System, Loinc Axis 4; T4 Dna Ligase; Techniques; Technology; Testing; Chromatin Immunoprecipitation; Cost; Cross-Link; Crosslink; Experiment; Experimental Research; Experimental Study; Gene Product; Genome Mutation; Genome Sequencing; Genome-Wide; Interest; Next Generation; Novel; Public Health Relevance; Reagent Testing; Research Study
