ChIP seq sample prep for Illumina

[athos]

Hi Everyone, I'm hoping someone can give some advice on ChIP enriched DNA. Does anyone try and quantify this by nanodrop/picogreen before starting the Illumina sample prep protocol (the protocol recommends starting with approximately 10ng DNA)? We have had difficulty in quantifying samples reliably. Is anyone doing ChIP seq regularly that can help?
Thanks.

[Chipper]

Yesy, we do but it does not tell you much since you do not know what is contaminaition and or background DNA. Usually PCR or qPCR will be more informative, if you have a good positive control.

[athos]

thanks Chipper. Can I just ask - do you normally follow the Illumina sample prep protocol as is, or do you ever do the size selection after the enrichment PCR?
Cheers.

[Chipper]

Hi, I have not done the sample prep myself and not seen the protocol but I thought size selection was supposed to be after enrichment as it makes more sense to me. Otherwise the risk of contamination by ladders or other saples increases and you will probably lose a lot of fragments during size selection. This protocol uses amplification first: http://www.genomecenter.ucdavis.edu/...brary_prep.pdf

[cjohns]

Here is what I found on our Chip-Seq:

Neither Nano-Drop nor bioanalyzer nor Pico Green quantitation of our IP products agree so we stopped trying to quantitate them. We use 1/3 of the IP product for the sample prep protocol (the same amount we used for Chip-chip and it gives us good results)

The gel purification step is much trickier than their protocol states. If you run the gel for 60 min as they state and cut out 200+/- 25 bp you will get a huge amount of adapter complexes in your final sample that will show up as a big peak at about 120bp on bioanalyzer results after amplification. They will also make up about 50% of your sequences on the machine. What we do is run a much longer gel for at slow speeds for 5+hours and cut out 180-300 bp and then continue with protocol as is.

Amplifying after size selection has not led to any perceivable contamination by ladders (of course, leave empty lanes between samples and ladders).

[Chipper]

Hi,
thanks for the heads up on the gel purification. How many cells do you generally use? I ment to say that contamination can be an issue if you dont have the adapters on when running the gel, but it only a few thousand such reads in our runs so not a real problem.

[jawdekar]

ChIP-Seq help

hi,

can anyone tell me if I can enrich the adapter-modified DNA fragments by PCR first followed by the size selection of the library. This is because I am starting will approximately 10ng of ChIP'ed DNA and it seems unlikely that I can visualize 10ng on a gel for me to excise DNA in the ~200bp range.

thanks

[tomv]

You can definitely gel purify after amplification. This is what we have done for a long time. We are just now testing whether its better to do it before, after or before AND after but the results are not in yet. I'd be interested to know if anyone else has tested this.

[alere]

ChIP seq enrichment

Hi everyone,
I'm preparing the ChIP enriched DNA for the ChIP seq.
I'm wondering if is possible (and better) do the size selection directly on the ChIP sample before the amplification step as the ILLUMINA protocol recommend. Is possible to see any band on a 2% gel loading the ChIP from 20x 10 6 cells?? In this case the adaptor ligation and the library amplification step will more specific leading to an effective 200bp long fragment enrichment.
Thanks.

[captainentropy]

athos, I agree with cjohns comment. I use Qubit for what I suspect will be really low concentration samples and Nanodrop for post-PCR quantitation. I've compared Qubit and Nanodrop on low [DNA] samples and they're usually close in numbers but percentage-wise they aren't even close (eg. Qubit says 6 ng/ul, Nanodrop says 4 ng/ul). Also, the tech in the Bioanalyzer facility says not to trust the Bioanalyzer for quantitation purposes as it has ~20% CV!

jawdekar, yes, you can definitely amplify after the ligation step. However, be warned that you may get massive amplification of concatemerized adapters that weren't removed in the clean-up step (Qiagen minelute). Thus if you want to amplify at this point it's important to use the right amount of adapters in the ligation step. As the Illumina protocol is written, using a 1:10 dilution is typically waaaay too many adapters. I use 1:30 as does another person I know that gets beautiful libraries (and that's ChIPed DNA from flies so there is tons of DNA per ChIP to start with). Basically you don't want your sample DNA to be the limiting factor but too many unligated adapters are problematic as they often become the preferred template for amplification and ultimately sequencing.

[xenia.zhang]

Hi all,

I have a couple chipseq libraries that show really good qPCR enrichment both before and after library construction, but sequencing results show very low signal, which resembles some other libraries where antibody is not optimal, though I know the Ab I use is a very good one. Then I start to suspect I have low ChIP enriched DNA for library construction, and tried to quantify concentration on high sensitivity bioanalyzer DNA kit. Unfortunately, for most of them, I can't detect anything, though the kit claims to be able to detect as low as 5pg/ul.

I wonder whether anyone else has this problem of quantifying chip enriched DNA with bioanalyzer high sensitivity dna kit, even with good Chiped dna samples? Then I wouldn't worry too much. Or it's really problem of my sample?

Or does any of you have any experience in this similar good qPCR, bad sequencing situation?
Million thanks to all!



-Sherry

[xenia.zhang]

BTW, immunoprecipitation itself does work. About half of the peaks callled are real. The others are basically either background or 'peaks' around 30bp, which are false positives.

[captainentropy]

from what I've been told about the Bioanalyzer is that it's not very good for quantitation purposes. I use it just to see what my library looks like (is there a peak around 80 or 125 bp indicating adapter carryover). According to Illumina, even if you see nothing on the bioanalyzer you may be able to generate clusters for sequencing. Even when I've had a tiny peak on the bioanalyzer chromatogram the sequencing was successful. For quantitation I use either a Nanodrop or Qubit. Qubit is more sensitive at low DNA concentrations. I have not done qPCR on my samples but the guy who does my sequencing does qPCR (on my samples) and I've never been told there was a problem.

What exactly do you mean when you say "About half of the peaks callled are real." Are you saying that the half that are "real" passed QC and uniquely mapped to the genome? I assume you have a bioanalyzer chromatogram? Was there a big peak around 70 or 124 bp? If so that is due to adapter carryover in the size selection step. I had that happen a couple of times and one time much less than half the reads mapped.

[zhaoj]

Hi Sherry
I had that problem before, I suspect the quantity of DNA was low in my CHIP sample (hard to quantitate), so I combined several ChIP and worked fine in sequencing.

[xenia.zhang]

Hi zhaoj,

Thanks for your answer! That's also what i suspect. By combining several ChIP, do you combine at the final elution step, or you actually use more chromatin prep?

THanks.



-Sherry