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. 2006;1(2):729-48.
doi: 10.1038/nprot.2006.98.

Chromatin immunoprecipitation and microarray-based analysis of protein location

Tong Ihn Lee  1 , Sarah E Johnstone, Richard A Young
Affiliations

Chromatin immunoprecipitation and microarray-based analysis of protein location

Tong Ihn Lee et al. Nat Protoc. 2006.

Abstract

Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types.

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Figures

Figure 1
Figure 1
A sample timeline for the ChIP-Chip protocol. Individual steps are shown in white boxes. Steps that are typically performed on the same day are grouped by day, which is indicated in gray boxes. IP, immunoprecipitation; LM-PCR, ligation-mediated PCR.
Figure 2
Figure 2
Results of varying degrees of sonication on fragment size. A total of 2 × 108 crosslinked Jurkat cells were sonicated using the following conditions: Misonix 3000 sonicator with microtip; power 7; 24 cycles (30 s sonication, 90 s rest). Samples were removed at various times, crosslinks were reversed and DNA-purified, and run on a 2% agarose gel. Lanes with molecular weights are labeled M and lanes with sonicated material are labeled with the number of cycles of sonication. Sizes of molecular-mass markers are indicated. Twelve cycles of sonication provide a good degree of sonication. Four cycles of sonication results in undersonicated DNA. Note that sonication beyond 16 cycles results in little change in fragment size and is likely to result in oversonicated material.
Figure 3
Figure 3
2% agarose gel showing an example of input DNA, DNA after LMPCR amplification and labeled DNA. The lane with molecular-mass markers is labeled M. Input DNA is in Lane 1, LMPCR DNA is in Lane 2, labeled DNA is in Lane 3. Approximately 200–300 ng of DNA are loaded. Note that labeled DNA will fluoresce (and thus appear more abundant) when photographed with UV illumination due to the presence of dye.
Figure 4
Figure 4
Samples of hybridized arrays and scatterplots. (a) A portion of a hybridized and scanned array. The material is enriched for RNA polymerase II binding. Red features indicate enrichment. Bright green features are hybridization controls. Yellow and yellow-green features indicate no enrichment. (b) A plot showing processed results of the hybridization and scan shown in a. Signal intensities (log base 2) for the RNA polymerase II-enriched DNA are shown on the y-axis. Signal intensities (log base 2) for the input DNA are shown on the x-axis. Features outside the boundaries, indicated by the red lines, are significantly enriched at a threshold of P<0.001. Features that are below the diagonal and appear significantly enriched are usually control features or dust particles that have been mistakenly identified as features. Features that are above the diagonal and appear significantly enriched usually represent immuno-enriched features and indicate protein binding.
Figure 5
Figure 5
Examples of data from an array that are processed to identify genomic regions that are enriched for binding and verification by gene-specific PCR. (a) Another portion of the hybridized and scanned array shown in Figure 4. The material is enriched for RNA polymerase II binding. The circled feature is red, indicating enrichment. Yellow and yellow-green features indicate no enrichment. (b) A plot showing ratios of the RNA polymerase II enrichment. Enrichment is plotted on the y-axis. Genomic location is plotted on the x-axis. Probe location is shown by the blue circles. The circled probe represents the ratio seen for the circled feature shown in a. The red box indicates the region checked using gene-specific PCR to confirm enrichment in c. (c) Gene-specific PCR, confirming enrichment. Primers were used to PCR the genomic region, indicated by the red box in b for immunoprecipitated DNA and dilutions of input DNA. Molecular-mass markers are shown in the left-most lane, which is labeled M. Three-fold dilutions of input DNA are shown in lanes 1–3, as indicated. The immunoprecipitation (IP) is shown in the last lane. The amount of immunoprecipitate that is added to the PCR is equivalent to the last dilution of input DNA. (d) A portion of a hybridized and scanned array. The hybridized material is enriched for RNA polymerase II binding. The circled feature shows no enrichment. Yellow and yellow-green features indicate no enrichment. (e) A plot showing ratios of the RNA polymerase II enrichment. Enrichment is plotted on the y-axis. Genomic location is plotted on the x-axis. Probe location is shown by the blue circles. The circled probe represents the ratio seen for the circled feature shown in d. The red box indicates the region checked using gene-specific PCR to confirm enrichment in f. (f) Gene-specific PCR confirming lack of enrichment. Primers were used to PCR the genomic region, indicated by the red box in d for immunoprecipitated DNA and dilutions of input DNA. Molecular-mass markers are shown in the left-most lane, which is labeled M. Three-fold dilutions of input DNA are shown in lanes 1–3, as indicated. The immunoprecipitation is shown in the last lane. The amount of immunoprecipitate added to the PCR is equivalent to the last dilution of input DNA.
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