Hello. I generated a few ChIP-seq libraries using NEBNext® ChIP-Seq Library Prep kit. I've never had any problems in the past and always had nice libraries.
Recently, I came across one issue. The libraries still look alright on a DNA HS Bioanalyzer chip but they are really low concentrated (0.1nM) based on qPCR using the KAPA Library Quantification.
We perform a PCR enrichment of adaptor ligated DNA and then we run it on a gel for gel size selection. I've attached the image of one of the library just before gel size selection.
For troubleshooting purpose, I've also attached a Bioanalyzer profile of one of the library post-size selection and a gel I ran after qPCR quantification. The gel clearly shows how there is massive amount of bands around 120-130bp which corresponds to the adapters length.
Do you have any clue about what the issue can be?
Thanks
Federico
Hi Frederico,
You could perform qPCR of a few genes before library prep and after library prep, and compare the enrichment fold. If the enrichment fold didn't increase at all and the amount of DNA neither, you know that something is going wrong during your PCR enrichment step.
Good luck!
Do you have bioanalyzer results pre- and post-problems? Does the size of the peaks look similar? Is there an issue with your qPCR? You could measure the concentration using Qubit or similar and calculate concentration with insert size:
concentration (ng/ul) / (660 x insert size) X 1x10^6 = Molar concentration
Hello Federico,
I am a PhD student working on the histone H3 acetylation involvement in children encephalopathy due to high levels of bilirubin. I followed your question on Research Gate about your problem using the NEBNext® ChIP-Seq Library Prep kit. Did you solve it? Nowadays, I want to prepare ChIP-DNA library of my samples, but I obtained very low DNA concentration (as you reported in your question). I quantified the DNA using several methods, from spectrophotometer to pico green and Kapa kit, but the results were almost the same. I don't know why... I wrote to the technical support of the product, and after some of their suggestion I tried another time using the DNA control provided by the kit. The results were the same... it seems that the DNA disappears! Did you solve the problem? Do you have suggestion for me? I am very frustrated, so if you can hel me, thanks a lot in advance,
Regards
Eleonora
Hi Federico,
I have the same problem.
Have you figured out the issue? If not what quatification method you finally considered to load libraries for sequencing? It will be a great help.
Thanks
Mithun
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