Double peaks ChIP-seq libraries

[AlejandroRivera]

Hello guys I'm new at this forum and also new to the library preparation.

I'm using the NEB ultra II kit for library preparation of ChIP DNA, my problem is that after size selection and PCR purification I run my libraries in the HS Agilent chip to estimate the library size. What I found is that I have two peaks: one around 350bp (I selected for this size using ampure beads) and one unexpected peak around 1000bp. I read that this may be cause due to overamplification of my library. Beacause of this I tried an experiment where I used different number of cycles in the PCR (see file attached) but It doesn't matter the number of cylces the HMW DNA is still there.

Has anyone experienced any issue like this one?

Any input is welcome.

Cheers

Alejandro

[nucacidhunter]

Double peak seems to be there even in lowest cycle number. It could be caused by residual bead carry over from PCR purification.

[AlejandroRivera]

Thanks Nucacidhunter.

Turn out you were right. After some optimization with the size selection I manage to get rid of the big fragments.

Thanks a lot for your help