Defining In Vivo Targets of Nuclear Proteins by Chromatin Immunoprecipitation and Microarray Analysis

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Abstract
Table of Contents
Materials
Literature Cited

Abstract

This unit describes the combination of chromatin immunoprecipitation (ChIP) with microarray hybridization to determine the genome?wide occupancy profile of a DNA?associated protein. After conventional ChIP, the immunoprecipitated material is amplified by a two?step process involving primer extension followed by PCR in the presence of a modified nucleotide. The amplified DNA is fluorescently labeled in a reaction that couples dye to the modified nucleotide, and the labeled sample is hybridized to a microarray representing a complete genome. This method allows the study of a protein's pattern of DNA association across an entire genome with no need for prior knowledge of potential DNA targets.

Keywords: Chromatin immunoprecipitation; ChIP; microarray; amplification; PCR; dye coupling; protein?DNA interactions; ChIP?chip; ChIP?on?chip; genome?wide location; hybridization; whole?genome analysis

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Reagents and Solutions
Commentary
Literature Cited

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Materials

Basic Protocol 1:

Materials

De‐cross‐linked DNA samples: typically yeast DNA from chromatin immunoprecipitation and input DNA control; unit 21.3 )
5× Sequenase buffer (USB)
80 µM primer A: GTTTCCCAGTCACGATCNNNNNNNNN (see unit 2.11 for oligonucleotide synthesis)
10 mg/ml BSA
0.1 M DTT
10 mM dNTP mix: 10 mM each dATP, dCTP, dGPT, and dTTP (unit 3.4 )
13 U/µl Sequenase (USB) stock: dilute 1/10 immediately before use
10× PCR buffer (see recipe )
Amino‐allyl dNTP mix (see recipe )
100 µM primer B: GTTTCCCAGTCACGATC (see unit 2.11 for oligonucleotide synthesis)
5 U/µl Taq DNA polymerase
0.1 M sodium bicarbonate, pH 9.0
Cy3 or Cy5 monoreactive dye (Amersham): resuspend entire tube of Cy5 or Cy3 dye in 45 µl DMSO
3 M sodium acetate ( appendix 222 )
10 mg/ml sheared salmon sperm DNA (unit 20.1 )
2.5× array hybridization buffer (see recipe )
Array prehybridization buffer (see recipe )
95% ethanol
Array wash buffer (see recipe )
0.2× SSC ( appendix 222 )

QIAquick PCR purification kit (Qiagen)
Thermal cycler
MinElute PCR purification kit (Qiagen)
Microarrays (unit 22.1 )
Coplin jar or petri dish
Plastic racks for holding microscope slides
Plastic containers for washing microscope slides in holders
Tabletop centrifuge with adaptors suitable for holding plastic microscope slide racks (typically the same as the adaptors meant for holding 96‐well plates)
95°C heating block or water bath
LifterSlip coverslips for microarrays (Erie Scientific Company)
Rubber cement
Humidified chamber (see recipe )
45°C oven
Platform shaker

Additional reagents and equipment for PCR amplification (unit 15.1 ) and separation of small DNA fragments by agarose gel electrophoresis (unit 2.5 )

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Literature Cited

Literature Cited
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