1. Cells (at least 1 x 10⁷) are chemically fixed (e.g. formaldehyde) to crosslink protein to DNA.
2. Cells are lysed with either a hypotonic buffer or mechanical means to release nuclei.
3. After harvesting by centrifugation, the nuclei are sonicated to shear DNA into small fragments (200 - 1,000bp).
4. Antibodies against the protein of interest are used to immunoprecipitate the protein-DNA complex.
5. The immune complexes are isolated by the use of agarose or magnetic beads.
6. Complexes are washed to remove nonspecific material, DNA is eluted from beads, cross-links are reversed by high salt and heat treatment, and protein/RNA is digested away.
7. Following purification, ethanol precipitation, and re-suspension of ChIP DNA, a small aliquot of DNA and PCR primers can be used to amplify a region where the protein of interest is known to bind.
8. After validating the enrichment of ChIP DNA, fragments are amplified by either whole genome or random priming methods. Experimental and total DNA samples are labeled using 9mer primers that have Cy3 and Cy5 dyes attached and Klenow added.
9. The labeled experimental IP and total DNAs are co-hybridized to the array for 16 - 20 hours, washed, and scanned.
10. Array images are used for data extraction as pair files; genomic feature format (GFF) files are then produced for visualization of scaled log₂-ratio data. The intensity ratio of immunopreciptated to total DNA (not taken through immunoprecipitation steps) is plotted versus genomic position to identify regions where increased signal (i.e. DNA fragment enrichment) is observed relative to the control sample.
11. Peak files (.gff) identifying statistically significant binding/modification sites can be generated from the scaled log₂-ratio data, and peaks can be mapped to the transcription start site of each gene.

Before embarking on a ChIP-chip experiment to analyze transcriptional regulation on a genome-wide scale, you need to optimize several experimental conditions:

• Sonication of Fixed Cells – DNA fragment size needs to be within a 200 - 1,000bp range for a successful experiment. Overshearing DNA fragments (<200bp) will make the post-IP amplification step difficult, and consequently there will not be sufficient yields for the labeling reaction. Undershearing of DNA will result in inefficient immunoprecipitation.
• Antibody Qualification – Not all antibodies can effectively immunoprecipitate protein-DNA complexes. Therefore, you need to use a ChIP-qualified antibody or empirically determine which antibodies are effective. Since the antibody must bind to a DNA-bound, cross-linked protein, the demonstration of target-specific signal in Western blot or gel-shift assay is necessary but not sufficient in determining the suitability of a given antiserum or monoclonal antibody for ChIP-chip use. The easiest solution is to select a commercially available antibody already successfully used for this application. Click here for an extensive listing of ChIP-chip validated antibodies using Roche NimbleGen arrays.
• Antibody Titration – Since the affinity and avidity of antibodies can vary, the amount of antibody in each ChIP-chip reaction needs to be empirically determined.
• PCR amplification of ChIP DNA – To be truly confident that the antibody was successful in immunoprecipitating the protein-DNA complex, PCR amplification on a known binding-site region for that protein will need to be performed using either conventional PCR methods followed by agarose gel electrophoresis or by quantitative PCR.

Having the proper experimental controls is also an important aspect of any ChIP-chip experiment. Listed below are common controls that researchers are using in their experiments:

• Nonspecific IgG antibodies – The most common type of negative control involves adding antibodies that do not recognize a specific epitope, for example pre-immune serum or IgG. A potential pitfall is that since the antibodies do not immunoprecipitate effectively, the nonspecific DNA yield is often extremely low. Hence, the hybridization tends to be much noisier and can result in many false positives due to amplification of trace amounts of nonspecific DNA. Alternatively, the primary antibody can be omitted (i.e. no antibody control).
• Protein deficient cell line – Several upstream applications, such as target deletion or siRNA, can be used to perturb the expression of the protein of interest, hence decreasing the amount of immunoprecipitated material. Alternatively, a cell line that does not express the protein of interest could be used as a negative control.
• Tag specific antibodies – If your protein of interest has a tag, such as GST or GFP, you may want to consider using antibodies against them in cells that do and do not express this tagged protein of interest.
• Uncrosslinked chromatin – Another negative control worth considering is the omission of the chemical cross-linking step prior to immunoprecipitation. The absence of the chemical cross-linking step provides one measure of the potential false positive peaks originating from non-specific binding of chromatin to immunoprecipitation reagents or solid phase (beads, magnetic particles, etc.) used in the ChIP procedure.

A typical ChIP yields approximately 10 - 100ng DNA, far less than needed for subsequent labeling reactions and hybridization to the array. Hence, ChIP DNA (and input DNA) must be amplified using one of the three methods to obtain adequate yields. Multiple cycles can be performed to achieve the desired amount of amplification product. Regardless of the method selected, amplified NimbleGen recommends the QIAquick PCR Purification Kit (Qiagen #28104)

• Whole Genome Amplification (WGA) – Genomic ChIP and input fragments are converted to PCR-amplifiable OmniPlex™ Library (Rubicon Genomics, Inc.) molecules flanked by universal priming sites. The library is then PCR amplified using universal primers and a limited number of cycles. NimbleGen recommends the WGA Kit (Sigma #WGA2-50RXN). The WGA protocol used by the Farnham lab at University of California - Davis can be accessed at genomecenter.ucdavis.edu/farnham/pdf/8-18-06WGA.pdf
• Ligation Mediated-Polymerase Chain Reaction (LM-PCR) – Unidirectional linkers are ligated to blunt-ended ChIP and input fragments and PCR amplified. Refer to www.chiponchip.org/protocol_itm3.html for a detailed LM-PCR protocol.
• T7 Amplification – Double-stranded ChIP and input DNA starting material is tailed on the 3' end of each strand to generate a 20 - 40bp polyT tail with a terminal dideoxycytidine base. A T7-(A)18B anchored primer adaptor is annealed to the polyT tail of each template strand. During second-strand synthesis, a Klenow fragment of DNA Polymerase I removes the excess bases from the tail overhang via its 3' - 5' exonuclease activity and extends from the primer to produce the second strand. This process results in two double-stranded DNAs identical to the original template, except that each has a T7 promoter at a different end. The product of second-strand synthesis is used as template in an in vitro transcription reaction. To generate DNA probes for labeling and hybridization, amplified RNA is reverse transcribed. Go to TLAD: DNA Linear Amplification - Web Supplement for protocol and FAQ regarding T7-based linear amplification of genomic DNA.

Quality Control of Amplification Methods

After amplifying ChIP and input samples, DNA should be run on an Agilent Bioanalyzer or agarose gel to analyze the size and quality of DNA fragments. The size of ChIP DNA fragments should be between 100 - 2,000bp with the majority of fragments between 200 - 1,000bp. Also, the DNA should appear as a smear, as opposed to several distinct bands. This banding results in noisy ChIP-chip data that is difficult to interpret. The figure on the right is an example of high-quality (smear) and proper fragment size ChIP DNA, as amplified by LM-PCR (lanes 4 and 5). Also shown are poor-quality (banding) LM-PCR ChIP samples that are too small to efficiently label (<200bp, lanes 2 and 3).