I am doing Chip-seq, but at the end of the experiment I have obtained a clear DNA background for my negative samples but absolutely no DNA from my tested samples. I think there might be something wrong with my de-crosslinking step but I don't know where or how I can make sure if my de-cross linking step works. Any idea? My chip-seq protocol was used based on this journal 'Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli'
Hi!
Test the crosslinking:
Compare Formaldehyde (FA) treated vs untreated DNA after sonication and purification on a gel - uncrosslinked DNA will be shredded in no time.
I crosslink in 1xPBS with 1% final FA for 10 min at room temp.
I always do an over night decrosslinking at 55 C in parallel with a Proteinase K digest. This works all fine. Over night decrosslinking this this condition is very efficient. I have no idea how to test this - but I wouldnt even bother to question this step.
I don't get really get the pic you send, but just to be clear - you won't be able to see a DNA smear after ChIP on a regular agarose/EtBr gel - unless you didn't wash the beads properly ;-)
Did you do a PCR on a positive and negative control region? We measure the DNA yield after ChIP with the Pico Green Kit on a fluorescence-based NanoDrop machine http://www.nanodrop.com/Library/PicoGreen%20-%20dsDNA%20protocol.pdf
Hope this helps.
Cheers,
M
thanks markus!
i cant do a PCR to test for my DNA because I dont know what region of DNA my protein binds to.. a lot of people have been suggestion bioanalyzer to detect if i have DNA to send in for sequencing..but i wonder if there is any other method available to verify if i have DNA as end result of my experiment?
Maybe some high sensitivity DNA chip for the bioanalyzer might help you, but I would try to go for the pico measurement mentioned above. Alternatively, you can always amplify some neg. control region - there is always some background signal in your ChIP, which will amplify. You can try an active promoter such as Tbp - I mostly use a gene desert region too. Alternatively, prepare a library for illumina sequencing with the NEB ultra kit. Do the preamplification - if you monitor this in real time PCR, such as the KAPA kit, you should get a proper signal after 8 - 15 cycles of amplification. You could also do an endpoint amplification with some of the library with 30 cycles and check this by gel/bioanalyzer/nano drop.
You should use Qubit for measuring ChIP DNA as nanodrop and bioanalyzer give you overestimation of DNA concentration. Markus is right that chromatin from crossliinked cells takes longer time than shearing uncrosslinked cells. Illumina micro Chip is great for library preparation when you have very little amount of DNA.
I hope this helps. Good Luck!
Dear Liyana,
You can reversecrosslink overnight at 65 C and then perform the Proteinase K treatment.
You can try to use SYBR Gold for the visualization. It is going to increase the sensitivity 100 fold.
The best option you have is Qubit HS dNA kit probably.
Finally, you can perform a library and then quantify the library by qPCR using commercial standards for the quantification. I personally recommend KAPA library quantification kit.
Good luck!
Hi Liyana.
I completely understand your situation and tried my best to answer ways to clear off your doubts. I hope this helps.
If your formaldehyde is fresh and you are using in appropriate concentration, than I do not find any reason to doubt it. However, You can do mini column purification for your crosslinked DNA in question. If it is un-crosslinked, you will be able to elute DNA from the column membrane, but if its in DNA-protein complex (crosslinked) then everything will be stuck in the membrane and you wont be able to elute it or wash it off (untill unless reverse-crosslinking is performed correctly), and then take 260/280nm reading.
And now, as far as reverse-crosslinking is concerned, if your reverse crosslinking has not worked then, you will have hard time resurrecting DNA even after phenol chorloform isolation. you might end up throwing of Protein-DNA complex. Most of the reverse-crosslinking is taken care by proteinase K digestion. However other methods like high salt treatment (500mM-1M NaCl) along with or prior to protienase K digestion helps to break tough DNA-Proteinase complex. Addition of 1%SDS also helps. Even harsher methods use Guanidinium hydrochloride to dissolve toughest of protein interactions. You can try these, if you want to make sure that reverse-crosslinking has worked. You might have to optimise it, if thats the issue you are currently struggling with.
I hope this answer helps in you solving you crossllinking-reverse-crosslinking doubts. but I am sure that ChIP-protocols have so many other steps to ponder upon.
All the Best!
I had to check that when I adjusted ChIP protocol for tomato plants.
Shortly you can check it by performing Phenol-chlorophorm (PIC) extractions. If the DNA is croslinked with proteins you won´t be able to find it in the arqueous phase whereas non croslinked DNA will be found mostly in the arqueous phase. So performing PIC extractions you can see if your DNA is crosslnked or not.
Here you can find how it can be done https://www.researchgate.net/publication/43097744_Protocol_fine-tuning_of_a_Chromatin_Immunoprecipitation_%28ChIP%29_protocol_in_tomato?ev=prf_pub
I hope you find this information usefull
Article Protocol: Fine-tuning of a Chromatin Immunoprecipitation (Ch...
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