Double band in ChIP-seq lib prep for Illumina

[zhaoj]

I hope somebody can help me out here.

I often get double band in ChIP-seq lib prep for Illumina platform. One peak is about 200bp, and additional peak around 260bp. This happens often, but not all the time, really annoying. Does any body have clues?

Could it be bad RNAase or Proteinase K on treating the ChIP samples? Because a recent exp has one old sample working fine (single 200bp peak), but not the new sample.

Or too much adaptor in the library prep?

Thanks in advance for your help!

Jh

[mountainogre]

Hi Jh,
Did you make any progress solving this problem?
I have just observed the same thing and i don't know why.
Have you seen this blog post: http://dnasizing.com/?p=105

[zhaoj]

Not yet, but suspect
1. RNA leftover
2. inappropriate ratio of adaptor to DNA
Frustrated, but I do not see it everytime, hope there will be an answer.

Good luck with your experiment!

[CC_seqanswers]

May I ask what sizing technique in your case?

Not yet, but suspect
1. RNA leftover
2. inappropriate ratio of adaptor to DNA
Frustrated, but I do not see it everytime, hope there will be an answer.

Good luck with your experiment!

[zhaoj]

I tried cutting out from agarose gel

[mountainogre]

And in my cases I size select with the e-Gel system. I aimed for the 180-200bp range.
My situation was similar to jH, in my experiment 2 from 8 libraries had the strange appearance of a stronger band around 260bp, whereas the Bioanalyzer showed appropriate 180bp band only for the other 6 samples.
I suspect some problem with over-amplification, since the two libraries with the extra band were the ones with the most DNA at the beginning of the protocol.

[zhaoj]

How much adaptor do you use in each reaction?
I use 1ul of 10 times diluted from Illumina.

[mountainogre]

I use the same. I should also mention that I am doing ChIPseq, not RNAseq, so things could be a little different.
I think I will repeat the prep with less starting material and see if that solves the problem.
In this case I used 13-15ng on ChIPPed DNA, I think i will try <10ng.

[pmiguel]

All,
I think the extra peaks phenomenon can derive from a number of different sources. I listed ones that occurred to me here:

http://seqanswers.com/forums/showpost.php?p=45116&postcount=19

One odd thing in this thread -- the idea that the slower migrating peak is the single stranded one seems contrary to my intuition. But, for Agilent chips I think it is the case. For details see:

http://seqanswers.com/forums/showthread.php?t=12852

http://seqanswers.com/forums/showthread.php?t=12501

I am just wondering are there resources that state this? The fact that the link upthread is implying that this is the case, suggests there must be. But I had not heard it previously.

--
Phillip