As part of my study I have to present on a method of discerning binding sites of proteins to DNA, for which I have looked at ChIP-seq. This is a brief overview of what I have so far, and was wondering if there are any particularly glaring omissions that I have made so far.
Cells treated with formaldehyde to cross-link proteins such as transcription >factors (i.e. HIF) covalently to DNA and incubated for 2-30 minutes. After >incubation glycine is added to terminate the cross linking and dissipate the >formaldehyde.
Cells are lysed, and sonication (application of sound waves to agitate a >sample) is used to fragment the DNA into sizes between 200-1000 base pairs.
HIF–DNA complexes are selectively immunoprecipitated by using antibodies >specific to the HIF protein of interest, complexed with a matrix of either >agrose or superparamagnetic beads.
HIF-DNA-antibody-matrix complexes are isolated through the use of either a >centrifuge or magnetic field (depending on the matrix employed). Several >washings take place to further purify the sample.
Cross-linking of the protein to the DNA is reversed by way of heating, >separating the DNA and protein-antibody-matrix complex. Proteinase K is used to >digest proteins, leaving only the DNA.
Oligonucleotide adaptors (primers) are then added to the small stretches of DNA >that were bound to the HIF to enable rapid simultaneous sequencing.
DNA sequences are then mapped to the reference genome to determine the binding loci.
I would also like to discuss controls that are used in these procedures. So far I have found limited mention of negative controls, positive and negative control loci, non-template control and positive control antibody, but am having trouble discerning what any of this actually is referring to.
I would seriously appreciate any help available on this topic!