Failure for Chip-seq library preparation

[binlu]

Hi all,

I am hoping someone would give me real help, asking for true help. I am facing big problem making chip seq library by illumina tru-seq kit. My target gene is TF, I fused 3Xflag to my protein, used 50 million cells for CHIP. Before library preparation I did Q-pcr to validate whether my chip is working, my find was that my positive region was enriched by CHIP. But I always failed in making library, very low concentration of my library. Does anyone know how to improve in my case? Need help. Thanks so much.

[jdk787]

How are you fragmenting your DNA, and have you checked (Bioanalyzer) to make sure that it looks right before starting your library prep?

[binlu]

I use bioruptor machine to sonicate my DNA which was optimized by my colleague, this protocol works fine for him. I have not checked the size of DNA preparation, people says it too low to detect by bioanalyzer? I was also worried about the distribution of my chip-ed DNA, since protocol needs to cut size rang of 250-300 which is equal to DNA of 120-200 before adapter ligation, if there is few DNA within this range, it is impossible for me to get good library, should I understand like this?

[huguesparri]

As sonication is performed before Immuno precipitation, you should be able to see your sonication profile on a bioanalyzer, especially when starting from 50 millions cells.
I advise to quantify you sample using microfluorimetry (Qubit for example) in order to determine what kind of chip you should use on the bioanalyzer (High sensitivity vs DNA 1000).
The other usefull information is how much material you start with to build your library and how did you quantify it?
Once again, microfluorimetry is mandatory. Do not use nanodrop!!!
If you are using the Truseq ChIP sample prep kit, I would not advice to start with less than 5ng per reaction, 10ng being really comfortable.
I also advise you to perform your PCR reaction before your size selection. It gives much better results in terms of sequence diversity.
Finally, for the size selection step, we usually size select depending on the maximum intensity of the signal once we see it on gel (or after Bioanalyzer if you size select with an automaton).
Good luck.

Hugues

[jdk787]

Agree with all of the points Hugues made.

The most common cause of ChIP-Seq library prep failure that I see is that the DNA going in to the prep is either not fragmented to a compatible size, or not quantified accurately so following Hugues recommendations should take care of that.

[binlu]

Thanks both of you, really appreciate. I am considering to optimize the sanitation condition.

what size of DNA should I start after sonication?

[binlu]

We followed the Illumina tru-seq protocol,
1, End repairs chip-ed DNA and purify with mini elute column
2, Add A overhang followed by adapter ligation and then do Ampure beads purification.
3, Size select with 2% DNA gel, and purify with mini elute column.
4, PCR enrich adapter ligated DNA by 15 cycles.
5 Use Ampure beads to purify final product.
I saw people doing PCR before size selection, can you share with me the protocol you are using?

[pmiguel]

I think the problem with the Illumina ChIP kit is that it uses an agarose gel extraction -- pretty much a guarantee that a large percentage of the library will be lost. I would recommend looking for a kit that doesn't use a gel purification. For example, Bioo has one. Or just replace the agarose gel, etc. with an an ampure lower size cut.

--
Phillip

[jdk787]

We followed the Illumina tru-seq protocol,
1, End repairs chip-ed DNA and purify with mini elute column
2, Add A overhang followed by adapter ligation and then do Ampure beads purification.
3, Size select with 2% DNA gel, and purify with mini elute column.
4, PCR enrich adapter ligated DNA by 15 cycles.
5 Use Ampure beads to purify final product.
I saw people doing PCR before size selection, can you share with me the protocol you are using?

You could do PCR first and then the gel cut. This would allow you to see if there is any DNA present, and if it is the correct size for what you are selecting for.

[huguesparri]

I think the problem with the Illumina ChIP kit is that it uses an agarose gel extraction -- pretty much a guarantee that a large percentage of the library will be lost. I would recommend looking for a kit that doesn't use a gel purification. For example, Bioo has one. Or just replace the agarose gel, etc. with an an ampure lower size cut.

--
Phillip

It's only an issue if you're size selecting before the PCR. At least, that's what we figured out from our experiments.
If you perform the PCR before the size selection, even if you loose material during the extraction, you will still have enough for several HiSeq lanes. And I don't think anybody needs that amount of data for ChIPseq

In our lab, we decided to perform the size selection on a Pippin HT. It saves us time and gives good reproductibility.

[binlu]

I have the problem as Phillip mentioned, I did Q-PCR foe chip-ed DNA, my input gets ct value about 25 cycle, my IP sample gets ct value about 28 or 29 for negative region, 22 for positive region. After library preparation, all my primers give me 32 or 34 Ct value even for input sample. it seems I lost most of my product. Does the length of primer affect the Q-PCR amplification, since my primer give me a product with length ~120?