Hi Steven,

I don't think that the DNA amount is the problem, I use about the same amount for a ChIP-qPCR sample. How do you measure your ChIPped DNA? On the Nanodrop or Picodrop? Does your sequencing facility measure the [DNA] before library preparation (I guess, they have to)? Depending on which system you use, you might get false values for your samples. Anyway, I also don't think that glycogen is the problem. Switching columns is for sure a good idea. I'm using the ChIP clean & concentrator columns from Zymoresearch (don't know, if they are available in RSA, though). They work pretty well for me (mostly even better than Qiagen columns) and are not that expensive.

Which histmark are you ChIPping for? Is it a rare one? Have you ever tried to pool 3-5 ChIP samples?

Let me know what you think and we can further discuss, if you like.

Best,

Kevin

Halo Steven,

I agree with Kevin, the amount of DNA is not the limiting factor.

In my experience, the enrichment of chiped material in qPCR can vary a lot depending on how you calculate (% input, fold over negative region, normalization to histone, etc.)

Primers and selected regions are also extremely important, many genomic regions are really impossible to detect! with standard condition. Try also many primers combination.

Glycogen is fairly neutral and phe/ChCl3 purification is usually ok if you remove well the phenol. The columns are also fine (but you loose some material...)

Hope this is helpful

good luck

alessandra

Hi Kevin and Alessandra

Thanks for the helpful comments. 

I measure ChIP DNA concentration using the Qubit HS dsDNA kit - apparently quite accurate. I get out about 2 ng (sometimes more depending on the mark) from several microgram Qubit-quantified chromatin. I notice you both work in medical fields, but I extract chromatin from woody tissues which is very difficult and gives lower yields compared to cell cultures. I usually pool 3 ChIP reactions for ChIP-seq, otherwise library complexity is compromised. I've prepared great libraries for H3K4me3 and H3K27me3 before - I'm repeating these for other tissues such as leaves now. I'm using validated primers (for a few targets) that have worked before, where I used amplified ChIP DNA. I don't do the amplification anymore because it induces too many duplicated reads.

Thanks for the suggestion to use Zymo columns, they are indeed available here. Hopefully that does the trick. I suspect there might be carry-over of phenolics from the plant tissues to my purified samples using standard purification.

How much DNA do you obtain from a typical ChIP, and how do you quantify the chromatin you put in? The literature is frustratingly vague on these details :P

Warm regards

Hey Steven,

I can imagine that it is a pain to get chromatin out of wood =), but at least you're (probably) not limited in the amount of material available. I'm looking at weak/very dynamic chromatin interactions and thus I sometimes have to pool up to 5 ChIP samples for ChIP-seq. The "typical" yield greatly varies, for histmark ChIPs I get tons of DNA (1 ChIP from 50 µg of input chromatin yields up to 80 ng, depending on the mark [more for K4me3, K9Ac and K27me3 and way less for K9me3], but this is also highly dependent on the antibody and the amount of it that you use, unfortunately). For other proteins I pool the samples and struggle to scrape together the 5-10 ng for ChIP-seq. 

Yes, I know, the literature is not very helpful in these issues. I know one case, where people use 50-100 µg of chromatin and throw in 15 µg of purified antibody (!). 

Addressing your original question: for me it's the same, reproducing with ChIP-qPCR what I see in ChIP-seq, is a lot of tinkering around. At what resolution do you look at your ChIP-seq data? Have you tried to "cover" the area, let's say 1kb, with several primer pairs? Maybe the mark is not exactly at the location suggestion by the ChIP-seq data.

Good luck! (And I wanna see the publication! ;)

Cheers,

Kevin