[Hamid]

Hi Khannak,

It seems that you are already working with chromatin material that has been sheared based on the bioanalyzer trace you shared. The problem is that almost 50% of the sheared material is over 700bp.
The questions I have are:
1. How was the chromatin sheared initially?
2. In what type of a buffer was the chromatin sheared?
3. In what type of buffer was the IP carried out?

Thank you

Hamid

[khannak85]

Sorry, but wouldn't shearing of DNA post IP in a ChIP-Seq experiment yield wide and unfocused peaks? I mean, you would end up sequencing fragments not directly binding your factor.

After IP, you should only pull-down specific DNA. Once you have that, for NextGen sequencing, we shear it further to obtain a median size range of 200bp and then add adapters. So now all your target DNA is within the size selection range and there would be minimum loss due to size selection.

[AndersHaakon]

Well, it is a ChIP-Seq project. So as a part of the ChIP protcol, DNA is sheared. But it is usually not exactly the size we want to go for library construction. Therefore, we sonicate it again to bring it down to a median size of 200bp.

Sorry, but wouldn't shearing of DNA post IP in a ChIP-Seq experiment yield wide and unfocused peaks? I mean, you would end up sequencing fragments not directly binding your factor.

[khannak85]

But how much of your material was in the size range that is getting selected? Thats why I was asking about whether you manually integrated your traces at any point.

If the shearing was ok. I would expect atleast 80% of the DNA to be in that size range. Since I only sheared 25ng of DNA. The concentration was too low to be analyzed on the bioanalyzer chip

[khannak85]

I've got an update on this issue.

On my first post I mentioned that I cleaned up the DNA using 1.8X AMpure but still no luck. So I tired cleaning it up with Qiagen PCR Purification kit and then made library. It was successful.
Although I am glad that Qiagen column worked, but it posed a new question in my head. Why did Qiagen column worked better than 1.8X AMPure? I know both have a lower size cut off of around 100bp and both should technically get rid off all the salt and contaminants

[DMO]

But how much of your material was in the size range that is getting selected? Thats why I was asking about whether you manually integrated your traces at any point.

[khannak85]

Now I am really confused. Why do you have small DNA fragments prior to shearing?

Well, it is a ChIP-Seq project. So as a part of the ChIP protcol, DNA is sheared. But it is usually not exactly the size we want to go for library construction. Therefore, we sonicate it again to bring it down to a median size of 200bp.

[ETHANol]

I do not have an image for Post-Shear, since the DNA was too diluted to be analyzed on a bioanalyzer. But I have attached the DNA profile pre-sonnication and the final library profile.

Now I am really confused. Why do you have small DNA fragments prior to shearing?

[DMO]

Did you do manual integration of your bioanalyzer run to see how much of your 25ng is in the 200-400bp range?

[khannak85]

I have successfully used SPRI-TE with as little as 10 ng of DNA for making ChIP-Seq libraries. As a matter of fact, this project has 12 samples with the lowest amount being 9 ng. All samples worked fine except the 4 ChIP input samples which are the cleanest.

I did think about the option of making them manually, but without a possible reason for these faliures, I feel manually library prep is going to yield the same results

[DMO]

I would recommend prepping manually as the SPRI-TE loses a LOT of DNA in its prep. I would only recommend using the SPRI-TE with 100ng+ of starting material. I regularly prep with 10ng (sometimes less) manually with no issues. I do not use the Illumina kits, but I do know others who do so with success.

[khannak85]

I do not have an image for Post-Shear, since the DNA was too diluted to be analyzed on a bioanalyzer. But I have attached the DNA profile pre-sonnication and the final library profile.

Thanks for the help.

[ETHANol]

Can you post an image of your sheared input DNA.

[khannak85]

Well, I'm trying to make a ChIP-Seq Library from 25ng of DNA sheared down to a median size range of 200 bp using covaris. Library construction is performed on Beckman Coulter SPRIworks station using their System I reagent kit (for Illumina) and Bioo Scientific adapters with a 200-400bp size selection.

The final product is amplified using KAPA HiFi and 15 cycles of PCR enrichment. Amplified library was cleaned up using 0.9X AMPure and analyzed on Bioanalyzer High Senstivity Chip.

[ETHANol]

Your going to have to give a much much more detailed explanation of what you are doing for anyone to be able to help you. So if you want an answer, spend some time and explain in detail what you are doing, including your exact protocol.

That being said, this is what I do and it works really well.

http://ethanomics.wordpress.com/chip-seq-library-construction-using-the-illumina-truseq-adapters/