Cells for chromatin immunoprecipitation and methods for making
The present invention provides isolated, non-viable bacterial cells comprising a plurality of nucleic acid crosslinks, methods for making said cells, and methods for using said cells to isolate DNA-protein complexes. In particular, the nucleic acids of the cells are crosslinked by contacting the cells with a furocoumarin compound and ultraviolet light.
1. A plurality of isolated, heat-killed, chromosomal DNA interstrand-crosslinked, cell wall protein-crosslinked, protein A-positive Staphylococcus cells comprising a plurality of cell wall protein crosslinks and a plurality of chromosomal DNA interstrand crosslinks, wherein the protein A-positive Staphylococcus cells are heat-killed, each cell wall protein crosslink comprises a covalently-bound aldehyde crosslinker chosen from formaldehyde, paraformaldehyde, or glutaraldehyde, and each chromosomal DNA interstrand crosslink comprises a covalently-bound ultraviolet light-activated crosslinker chosen from psoralen, 8-methoxypsoralen, 4,5′,8-trimethylpsoralen, or 4′-aminomethyl-4,5′,8-trimethylpsoralen, wherein a plurality of DNA-protein complexes isolated using the plurality of isolated, heat-killed, chromosomal DNA interstrand-crosslinked, cell wall protein-crosslinked, protein A-positive Staphylococcus cells comprises Staphylococcus DNA that does not interfere with sequence-independent analysis of DNA in the plurality of DNA-protein complexes, and the plurality of DNA-protein complexes has reduced amplifiable Staphylococcus DNA as compared to the same DNA-protein complexes isolated using a plurality of heat-killed, cell wall protein-crosslinked, protein A-positive Staphylococcus cells that are devoid of chromosomal DNA interstrand crosslinks.
2. The plurality of isolated, heat-killed, chromosomal DNA interstrand-crosslinked, cell wall protein-crosslinked, protein A-positive Staphylococcus cells of claim 1 , wherein the plurality of chromosomal DNA interstrand crosslinks comprise 4′-aminomethyl-4,5′,8-trimethylpsoralen, and the protein A-positive Staphylococcus cells are Staphylococcus aureus Cowan strain 1 cells.
3. A method for isolating DNA-protein complexes, the method comprising:
a) contacting a plurality of DNA-protein-antibody complexes with the plurality of isolated, heat-killed, chromosomal DNA interstrand-crosslinked, cell wall protein-crosslinked, protein A-positive Staphylococcus cells as recited in claim 1 such that the plurality of isolated, heat-killed, chromosomal DNA interstrand-crosslinked, cell wall protein-crosslinked, protein A-positive Staphylococcus cells bind the plurality of DNA-protein-antibody complexes to form a plurality of protein A-positive Staphylococcus cell-bound DNA-protein-antibody complexes; and
b) contacting the plurality of protein A-positive Staphylococcus cell-bound DNA-protein-antibody complexes with an elution buffer to elute a plurality of isolated DNA-protein complexes, wherein the plurality of isolated DNA-protein complexes comprises Staphylococcus DNA that does not interfere with sequence-independent analysis of DNA in the plurality of isolated DNA-protein complexes, and the plurality of isolated DNA-protein complexes has an amount of amplifiable Staphylococcus DNA that is reduced by at least about 10,000 fold as compared to the same DNA-protein complexes isolated using a plurality of heat-killed, cell wall protein-crosslinked, protein A-positive Staphylococcus cells that are devoid of chromosomal DNA interstrand crosslinks.
4. The method of claim 3 , wherein the DNA in the plurality of isolated DNA-protein complexes isolated at step (b) is analyzed by a process selected from the group consisting of massively parallel DNA sequencing, sequencing-by-synthesis, sequencing-by-ligation, 454 pyrosequencing, paired-end tag sequencing, cluster amplification, bridge amplification, and PCR amplification.
5. The method of claim 3 , wherein the plurality of isolated, heat-killed, chromosomal DNA interstrand-crosslinked, cell wall protein-crosslinked, protein A-positive Staphylococcus cells are Staphylococcus aureus Cowan strain 1 cells, and the plurality of chromosomal DNA interstrand crosslinks comprise 4′-aminomethyl-4,5′,8-trimethylpsoralen.