I did a CHIP seq against a transcription factor, and to validate my results using CHIP PCR, I just take a look at the peak i have in CHIP seq.
For negative control, I design couples of primers in an area where no enrichment of my TF is observed, and for positive control, you can design primers in area where you have strong peak (strong enrichment of your TF). For me it was easy cause I had the datas from my own chip seq
If you don't, you can use CHIP seq data from other lab group (if your TF or histone mark as already been studied in other models), and use software like IGV (.bed file) or IGB (.wig file) to design your primers
Thanks Charly for your words of wisdom.
What is the main purpose of these softwares which you mentioned? Can I use them to find target genes and enrichment areas of my ChIP of a transcription factor or histone mark? Can we use UCSC browser to look for histone mark enrichment like for H3K4me3?
of course you can use UCSC.there are many data about the histone mark ChIP,just see the gene regulation colume and click to show the related histone modification data .But I the organism and cell type may be different from your interest type.But I think it's still of some reference function.
Yes, IGB and IGV are the two main softwares for manipulating CHIP seq Datas! They are very good for visualizing peaks and then design primers couples for PCR.
Usually in supp datas of a publication, you have attached files with the format .bed or .wig corresponding to the CHIP seq datas against TF or Histone marks
In IGB i open .wig files, this software is quite easy to use. Imagine you open a file corresponding to CHIP seq against H3K4me3, then you can look for any genes and visualize the peaks surrounding it (.wig) file with give peaks relative intensity, so that can you can see the relative enrichment between every region in the genome
In IGV, I open .bed files, for a given CHIP seq, this software will also give you the statistically relevant peaks so that you can design couples of primer in the surrounding region. In IGV is very useful because you can easily upload dozen of files from other CHIP seq datas, so that for a specific gene in a given cell type, you can see the enrichment of all the histone marks and TF of your choice...
I suggest you download these software if you want to get into CHIP seq, they are quite easy to use even for a debutant! For primer design i suggest to open .wig files in IGB, once you get the DNA sequence corresponding to the peak you want to study then you go on primer3 for primer design :)
i hope i was quite clear!
Thanks Qing for your quick help, but I could not find gene regulation column to look for the ChIP seq data. Could you please elaborate little bit of it?
If you are looking to confirm a successful ChIP experiment before sequencing, you can use qPCR with primers designed around probable regions of enrichment (positive control) and around potentially non-enriched regions (negative control). This method is however more easy to use for histone marks than for TF, since you can know more easily which regions will be enriched or not enriched for histone marks.
For example, for ChIP DNA specific of histones bearing the H3K4me3 post translational modification, you can design primers specific of the Transcription Start Sites of the most expressed genes as a positive control. Primers too far from the TSS can be used for negative control.
Be sure to also test input DNA in qPCR for normalization.
It is also wise to perform a first ChIP sequencing of ChIP DNA of interest, input DNA and ChIP IgG DNA with a low sequencing depth to test if your ChIP experiment was successful. IgG DNA is important to identify hyperChiPable regions while input DNA can be used as normalization in ChIP-seq to mitigate biases, notably during peak calling. Paired end sequencing can also be used to perform more precisely the peak calling since you'll not have to infer the length of DNA fragments (which is different from sequenced reads length).
We are just in the process of setting up a database for ChIP-primers.. www.chipprimers.com. If you have published and verified primers I'd be happy to include them, alongside your publication to cite.
Stefan, your database is a brilliant initiative! It's some kind of equivalent of RTprimerDB for RNA!
Once my CHIP experiments will be done, I will send you the list of the primer I used and the corresponding publication.
I'm keeping your website, I'll spread it to my CHIPping colleagues !
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