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Old 06-28-2013, 05:22 AM   #1
Location: Spain

Join Date: Jun 2013
Posts: 21
Question Chip and Input different sizes libraries (NEBNext ChIP-Seq Library)

Hi all!

I'm new here (and also in Chip-Seq!) and I would like share some questions with you.

We are preparing Chip-seq libraries for MiSeq, using NEBNext Chip-seq Library Prep Master Mix Set for Ilumina following the protocol with some modifications in the size selection of adaptor ligated DNA using AMPure XP Beads (we found a similar protocol that worked better for our samples , in order to get rid of the adaptor dimers).

After the amplification of the library by PCR, what we find is that the INPUT library has a correct size distribution but the CHIP library is different, having an important part of larger fragments, as you can see in the image I've attached here.

Of course, both come from the same sonicated sample and both have been processed and amplified together, with the same cycle number of amplification...

1. Any idea or explanation for this larger fragments( Maybe it's some issue related with the IP?)

2. Anyone has experience on sequencing this kind of library with those larger framents or knows how it could affect the sequencing process)?

Thanks a lot in advance!
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HelenaSC is offline   Reply With Quote
Old 06-22-2014, 05:49 PM   #2
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Location: australia

Join Date: Jun 2014
Posts: 1

we have a similar problem, did you ever solve your problem?
michaelftang is offline   Reply With Quote
Old 08-21-2014, 02:57 AM   #3
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Location: Cardiff

Join Date: Aug 2014
Posts: 1

Hi all,
I'm quite new in the ChIP-seq world, but I'm trying to optimise Ion ChIP-seq Library and I had similar problems with the bioanalyzer patterns, first I though it was something about the running, but after reading some answers from different forums and doing some more optimizations I'm almost sure that there are some important issues in the Chromatin immunoprecipitation:
1) you should have proper shearing of the Chromatin, so you should have the high concentration of DNA fragments between 100-200bp. That it's really important for the sequencing step.
2) When we're immunoprecipitating, longer fragments have higher affinity to the antibodies (I read that it can be due to the presence of more epitopes) so, for this reason in the IP sample from HelenaSC has larger fragments. I've solved this fact increasing the concentration of the antibody, we should saturate the sample so shorter fragments have also antibody available for IP.

I hope it helps,


Annac is offline   Reply With Quote

bioanalyzer, chip-seq, nebnext, size distribution

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