I am doing chip-seq. But the q-PCR result after establishment of the chip-seq library for the same loci is different from before chip-seq library. Who can tell me the potential reasons and how can I do to approve it?
In the Library preparation workflow a size selection is used, to get rid of free adaptors, that are not ligated to your sample, and to small and to large DNA fragments. So you loose some of your original material.
Hi, Dr. Martin:
Thank you very much for your answer! I have performed the size selection and chose the 200-500 bp fragment using the 2% E-gels (Invitrogen). Do you think there are other reasons?
Thanks again!
DNA extraction from a gel is not the best way to do size selction, you better use AmPure beads for this, these gives a much higher recovery and purity of your samples, you can repeat this size selection step to finally get better performance of your samples on a NGS platform..
Thanks a lot, Dr. Martin! I will try again according your suggestion.
Hi, Dr. Hongzhen:
As I know, PCR amplification may has different preferences to various sizes of ChIP-DNA fragments. Usually，ChIP-qPCR is just performed before the library preparation. The numbers of qPCR templates are changed before and after ChIP- Seq DNA library construction.
Another reason is that during the library preparation, you perform a preamplification step, where the signal to noise ratio increases. (I assume you see an increase in your fold enrichment calculations) This happens because fewer "background" molecules are present than target molecules, so with each pcr cycle, you are increasing the difference between the two. Hope this is clear.
Also, using the E-gels for library size selection is recommended by illumina, so I think that is pretty standard to do it that way.
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