[msheldon]

also another point, not related to the questions here: adding sybr into the gel mix (before it polymerizes) or post staining can have an effect of DNA running differently in agarose.

[Hamid]

Hi Everyone,

Sorry for being so late on this conversation, but we have seen this quite a bit, and I do have some input:
1. Both gel images shown have been severely overloaded. The gels are also run too fast. You can tell this by the fact the the sides of the lanes and running much further behind than the middle of the lanes. We run 1.5% agarose gels, and run the gel for 3-4 hours at 15-20 volts.
2. Bioanalyzer traces are log based, so a large distribution of higher molecular weigh fragments are compacted into a a much smaller area of the trace as compared to the smaller size fragments.
3. Sybr does effect the mobility of DNA in gels. It is suggested to add Sybr into the loading dye which is then mixed with the DNA samples prior to loading on a gel.
4. If staining with EthBr, then the gel should be post stained.
5. 1.5-2kb peaks noticed on bioanalyzer data when analyzing for chromatin sharing is due to over-cross linking of chromatin. Please take a look at the attached file in which we illustrate the appearance of the 1.5-2kb peak with an increase in cross linking. In our experience, this over-cross linked smear is not reduced further by shearing the chromatin longer.


Thank you

hamid

[Smriti]

Hi Hamid,

In line to your explanation of over-crosslinking of chromatin, I also did some preliminary work on chromatin shearing. I used Advance Analytical (Fragment Analyzer) and Agilent (Bioanalyzer) to analyze the sheared sample. Surprisingly, I see two populations of fragments, one at >1000bp and other at arnd 200bp. Do you think this is becof over-crosslinking or researchers do see this typical behavior with chromatin. Please explain.

Thanks,
Smriti

[Hamid]

Hi Smriti,

that is certainly because of over cross linking. we have found that in order to be maximize your chromatin shearing protocol, you must carry out a cross linking time course on the cells you are working with. Cross linking efficiency is certainly cell line dependent. We have customers who cross link certain cells for only 30 seconds because they lose their epitope if cross linked longer.
If you do a smear analysis on your bioanalyzer trace as we did, you will see that over cross linking will effect the percentage of fragments from 150-700bp. If you are doing ChIP-qPCR the percentage of fragments within the library preparation requirement of NGS systems does not make a difference, but if you are doing ChIP-seq it is important to maximize the percentage of fragments within the requirements of the library preparation of your NGS system.

Thank you

hamid

[ALive]

Hi everyone,

Wow I was happy to find this discussion this morning. I'm also getting huge peaks on the bioanalyzer after doing my chromatin immunoprecipitations, but it's a bit different to what you guys are describing:

I did a basic ChIP to prepare for sequencing (cross-linking with 1% formaldehyde for 10 minutes, sonication with a biorupter for 20 minutes to give fragments of about 200-500bp checked on an agarose gel).

I used magnetic beads with antibodies against histone modifications (plus controls). The input chromatin was 50% of that for the immunoprecipitations. The concentrations on the Qubit are between 0.4 ng/ul to 10 ng/ul. I checked the fragment sizes on a bioanalyzer (HS dsDNA kit) and saw peaks at ~150bp for my input chromatin. But the peaks for all of the immunoprecipitations were up around 3000bp. I'm lost and confused, and would really appreciate some thoughts on this problem

I've attached the graphs: on the left are two inputs, and on the right are two IPs. Also attached my gel after sonication (yes, I know now that it is overloaded and run too fast... ).

Thanks,

Ali

[ETHANol]

Your agarose gel is overloaded. You need to dilute the sample and run it again to get an idea of the size of your starting material. It will look substantially different after you dilute it and your fragmentation will look substantially less ideal. Running it longer will also help.

That being said it still does not explain the difference you seen on the Bioanalyzer between input and ChIPed DNA.

[pmiguel]

Some magnetic beads (eg SPRI/AMPure beads) evidently produce an artifact similar to the one you see. See, for example, this. (Slide 20).

And/or something co-purified with your DNA during your IP that is retarding its electrophoresis.

--
Phillip

[Hamid]

Hi Alive,

you have successfully sheared the chromatin to the 150bp, but in the process you have also destroyed the epitope. As a result when you IP, you are pulling down only the larger partially sheared and unsheared chromatin that contain the intact epitope. You can actually see the larger range in the bioanalyzer trace of your input. when you run the IP'd material on the bioanalyzer, you are noticing the larger fragment range which were IP'd as a peak from 1kb-10kb.
You are correct that your gel is overloaded, but so are your BioAnalyzer samples.

Thank you

Hamid

[Smriti]

Hi Hamid,

I have been following all the threads related to chromatin shearing on this forum. I kind of get similar result on IP showing larger fragments with high concentrations. Do you think higher concentration has relation to the larger fragments? I thought concentration of sample is independent of the size of fragments. Can you help me understanding this? Also, in case you can share some article that explains the terms like- peak calling, cross-correlation plots of the mapped reads and other QC metrics, it would be a great help.

Thanks,
Smriti

[ALive]

Hi again everyone,

Still no luck on my end.

Hamid - thanks for your advice. I was wondering about that. So I just have to sonicate for a shorter time and keep the fragments in a smear larger than 150 bp? Can anything else destroy epitopes?

Smriti - I ran two parallel IPs. Everything was identical except for the elution/reverse cross-linking steps (I used two different protocols). For one of the samples I got a high concentration on the Qubit (2 ng/ul) and enormous fragment sizes on the bioanalyzer. For the other I got a very low concentration (0.4 ng/ul) on the Qubit, but the enormous fragments were gone almost completely on the bioanalyzer. I am not knowledgeable at all about bioanalyzers, but it seems like (for my samples) the concentration is correlated with the size of fragments.

Thanks,

Ali

[spikey_hedgehog]

Hi Everyone,

This forum is fantastic! I am experiencing the same problem. The bioanalyzer has been giving me this 3000bp for my sonicated total input. So initially, I thought I had a problem with our sonication conditions. So I spent several weeks testing different conditions of soncation but all these tests resulted in the right DNA fragmentation sizes (200-600bp), as detected by the HS bioanlayzer chip.

However, when I did my real chip, it still gave me a large peak at 3000bp. I don't think the sonication procedure has damaged the epitope as the total input sample still gave me this 3000bp fragment. I tried to run all the DNA samples on the HS bioanalyzer chip at a similar DNA concentration (quantitated by the Qubit, at roughly 2-3ng/uL).

Considering that all my test runs have been consistently producing the right DNA sizes but not my real chip experiments- it is suggesting the problem exists during the IP step but what happens if the total input samples of my real chip experiment is also giving me a 3000bp? I dont know what else I could test?

Any advice? (I apologise for the desperate tone of my message!)

Thanks very much in advance
Hedgehog

[pmiguel]

See Hamid's post up-thread. He attributes this phenomenon to over-crosslinking.

--
Phillip

[Hamid]

Hi,

The problem with over cross linking of samples is that they will have to processed with higher amount of energy, and for a longer period of time to get the chromatin sheared to the smaller fragments. As a result most if not all of the epitope is destroyed during the process. You do get a very nice band showing great shearing, but the epitope will be destroyed. A great way to test it is to carry out a shearing time course, and then use 10-15ul of the lysates for each time point to do a western with the same antibody you will use for your IP. Use unsheared material as your control. You can easily then see the extent of damage to the epitope.
I have attached some data on the effects of over cross linking.
I hope it is helpful.

Thank you

hamid

[ALive]

Yes that definitely helps Hamid, thank you.

I'll do some westerns then and see what happens.

Ali

[shanshanzhou]

Yes that definitely helps Hamid, thank you.

I'll do some westerns then and see what happens.

Ali

Hi ALive,

Just wondering if you solved your problem with the large IP fragments. I have exactly the same problem with you, right shearing size, but large IP fragments. Thinking to do some westerns, but please let me know if you have any progress on that issue. Thanks so much!!

Best,
Shanshan