Hello, I'm new in the ChIP-seq field and had issues with my first experiment. I started with 5 ng of ChIP-DNA (as measured with Qubit, bioanalyser gave slightly different results)then followed the Illumina protocol with 18 cycles of PCR. When the sequence went back, I found >95% of duplication. The only peaks identified by macs seem to be PCR artifact. The antibody I used was validated by western blot and mass spec analysis after immunoprecipitation. So I know that the IP step is working.
I was considering re-doing the experiment increasing the amount of DNA to start with and maybe decreasing the number of PCR cycles.
Illumina recommends 5<<10 ng of DNA as starting material. Does anybody tried to start with more ie 30-50 ng? Looks to me as the only way to get better results
any help will be greatly appreciated. many thanks Olivier
