ChIP-Seq library - Microplex - bad profiles

[Valerie_Gaudin]

We have trouble with ChIP-seq library...
Does anybody have experience with diagenode MicroPlex Library Preparation kit v2 C05010014 (48 rxns, 48 indices)? We have bad bioanalyser profiles after library preparation with enrichment in very large fragments which seem to be concateners... ??? after denaturation and renaturation the profile come back to something more normal... very strange... we use low DNA quantity

so your expertise would be really appreciated!
Thanks a lot in advance!

[nucacidhunter]

The kit seems to be rebranded ThruPLEX DNA-seq kit. Your description indicates PCR overcycling. For a definitive answer posting library (before PCR purification) and input profiles would be helpful.

[Valerie_Gaudin]

Thanks I attached the aberrant profiles

[Valerie_Gaudin]

In attached file, a more complete description of the different steps..

[Diagenode]

ChIP-Seq library - Microplex

Hi Valerie,

Our tech support team have tried to call you today to discuss further your problem.
But in summary we believe that your library his overamplified. We recommand to amplify less cycles.
Let's discuss further on the phone...

Best regards,
Diagende's support team

[nucacidhunter]

With equivalent of this kit (ThruPLEX DNA-Seq) following PCR cycle number based on input gives good results. To avoid overcycling you can do 1 less cycle based on recommendation then run a sample of PCR on ScreenTape or other device. If you get <2ng/ul you can do extra cycles to increases yield by assuming doubling yield with each cycle. You should also see smaller peak at 60-70 bp in profile for primers. Lack of small peak indicates that reaction has run out of primers and all sorts of artefacts will show up. Glad that tech support is looking after the issue.

[Valerie_Gaudin]

Thanks for advices! checking the 60-70 bp peak is a good point!! We will do!
We also purified the sample before doing library with Ampure. Indeed for input DNA we got a peak on bioanalyser around 30-50 bp (steps1-2 in atached file) do you know whether that can affect library preparation or if there are better alternative to remore this small DNA fraction?
Thanks in advance!

[nucacidhunter]

Looking at #2 it seems that you have successfully removed the small peak (~50 bp) present in #1. That should not affect library prep and some protocols even call for size-selection in a narrow range. Purified input should be eluted in TE0.1 or Tris buffer because high EDTA adversely affects library prep with this kit.