ChIP-Seq combines chromatin immunoprecipitation and deep sequencing techniques to allow users to identify the binding sites of DNA-binding proteins of interest, including transcription factors and polymerases. ChIP-Seq is offered on our Illumina sequencing platform.
Protein A sepharose beads bind all antibodies and are a convenient way to separate antibody complexes from a mixture. Pansorbin® cells are heat-killed, formalin-fixed Staphylococcus aureus cells that have a coat of protein A. Both methods work equally well so which protocol to follow depends on user preference.
Formaldehyde is used as a cross-linking reagent because it is widely used to study bindingof specific proteins to DNA elements in intact cells. Also, cross-linking protein and DNA using formaldehyde is reversible (by heating the sample at 65°C for 5 hours).
An article in Biotechniques (Nov 2005, 39(5):715-725) by Nowak et al. have proposed a two-step cross-linking method that uses formaldehyde as well as disuccinimidyl glutarate (DSG), a cell-permeable, amine-reactive N-hydroxysuccinomide ester. This article suggests that DSG can stabilize multimeric protein-protein interactions prior to protein-DNA cross-linking with formaldehyde, thus increasing the sensitivity of the ChIP technique. The UHNMAC has not yet evaluated the use of DSG.
The samples are sonicated to shear the chromatin into lengths of approximately 1000 base pairs. In our lab, we typically sonicate samples in 6 intervals of 25 seconds each, with a 2 minute rest between intervals to prevent over-heating. On the Branson 150 Cell Disrupter, the power setting “3” is used.
Shearing efficiency is checked by running an aliquot of the sheared sample on a 1% agarose gel. DNA fragments should be around 1000 base pairs in length.
The sonicated sample is pre-cleared (prior to addition of specific antibody) with either Protein A Sepharose TE 50% slurry or Staph A cells in order to reduce non-specific background. If the sample is not pre-cleared, all antibody-protein complexes (not just the antibody-protein-of-interest complexes) would be precipitated and this may lead to background issues downstream.
The “no-antibody” negative control is produced by performing a parallel experiment without the addition of an antibody and therefore it will contain all chromatin species which bind non-specifically to the Protein A beads or Staph A cells. This control is important to use during PCR amplification. The "no-antibody" control can also be used as a control for the co-hybridisation of samples. When a spot fluoresces more in the antibody channel than the no-antibody channel, that DNA sequence is enriched in the antibody sample.
Total input control, as the name suggests, uses a fraction of the sample prior to the immunoprecipitation as a control sample. However, this control does not account for the non-specific binding of the StaphA cells (or beads) like the “no antibody” control does. The “no antibody” control sample is precipitated by the StaphA cells (or beads) but in the absence of antibody. The total input control can be used for comparison between treatments but the “no antibody” control eliminates false positives caused by non-specific proteins bound to the StaphA cells/beads.
Yeast tRNA is used as a blocking agent to block non-specific binding between Ab-protein-DNA complexes and Protein A sepharose beads.
RNA is digested using RNaseA to prevent non-specific amplification during PCR.
Proteinase K is used to digest the protein portion of the Ab-protein-DNA complexes so that the DNA (region of interest) can undergo PCR amplification.
Arabidopsis spots are not intended to be used for normalisation. The human and mouse CpG arrays also have Stratagene’s SpotReport™ Alien™ cDNA Array Validation System spotted on them, so users can use these for normalisation. An aliquot of the Arabidopsis control (plasmid containing the chlorophyll synthetase gene fragment), for use as a positive control, is available from the UHNMAC at no cost to users. The Alien™ cDNA can be purchased from Stratagene.
The appropriate PCR cycle number can be determined experimentally by setting up a PCR reaction and removing an aliquot of the PCR reaction at several intervals (15, 17, 19 and 21 cycles, for example). Each aliquot is then run on an agarose gel. The appropriate number of cycles is reached just prior to the cycle when the amount of PCR product on the gel plateaus, reaches saturation. For a pair of samples that will be analysed on the same array, the PCR cycle number should be the same for both samples.
Bovine serum albumin (BSA) is used as an adjuvant to increase the yield of PCR product.
Since Round B PCR amplification incorporates animo-allyl dUTP, any fluor or dye available in N-hydroxysuccinimide reactive form can be used.
No, ChIP samples must immediately be amplified with the round A protocol, followed immediately by round B amplification because the ChIP product is very unstable.
Ligation mediated (LM)-PCR involves attaching a unidirectional double-stranded oligo linker to sonicated DNA followed by PCR amplification.& LM-PCR does not take into account the different sizes of sonicated DNA, thus smaller fragments may be amplified more readily than larger fragments and very large fragments may not be amplified at all. This is the amplification method used in the Agilent ChIP platform.
Random priming involves hybridizing 6-8 mer oligos to sonicated DNA for PCR amplification of all DNA fragments of all sizes. In our experience, random priming results in arrays with lower background signal and better dynamic range compared with LM-PCR.
Magnetic beads, Protein A/G Sepharose beads and StaphA cells all used for the immunoprecipitation step.
The PMGC uses protocols using either Protein sepharose beads or StaphA cells as these methods are very good at binding the antibodies.
However, the protein beads and StaphA cells are not as specific as one would hope.
For this reason, we highly recommend using a “no antibody” control.
The magnetic beads are used for immunoprecipitation in the Agilent protocol. Although these beads have more specific binding capacity, they have less overall binding capacity and thus require more starting material (sample cells) than the previous two precipitation methods.