Dear all,
I am trying to set up ChIP-seq in our lab. My problem is that, following ChIP, the bioanalyser profile of my input and samples is very different. The input shows plenty of material in the 2-300bp range. However, all my ChIP samples show a peak at around 90bp and go to baseline after that (see attached).
Just to give you some info on the procedure I am using:
Starting with 7 x 10^7 cells (Drosophila Kc cells)
Fix with 1% formaldehyde for 10 minutes
Sonicate with the Bioruptor for 7 pulses (30secs ON 30secs OFF)
(I've optimised the number of pulses to make sure I get a smear of between 100bp and 1Kb when I look at an aliquot on a gel following reversal of crosslinking)
I block samples with Rabbit IgG for 3-5 hours and treat with the antibody for the TF I am studying O/N.
The beads are agarose G. I do the RNase, reversal of crosslinks and Proteinase K steps, and do Phenol-Chloroform extraction to purify using Glycoden and carrier tRNA.
I have also done this exact procedure with the positive control H3K4me3. qPCR indicated an enrichment of a positive site by 40-50% so I assumed the ChIP had worked. However, the bioanalyser profile is the same as for my samples with a peak at 90bp and nothing bigger.
If anyone has seen this kind of thing before and has any ideas I'd be really grateful.
Thank you!
I am trying to set up ChIP-seq in our lab. My problem is that, following ChIP, the bioanalyser profile of my input and samples is very different. The input shows plenty of material in the 2-300bp range. However, all my ChIP samples show a peak at around 90bp and go to baseline after that (see attached).
Just to give you some info on the procedure I am using:
Starting with 7 x 10^7 cells (Drosophila Kc cells)
Fix with 1% formaldehyde for 10 minutes
Sonicate with the Bioruptor for 7 pulses (30secs ON 30secs OFF)
(I've optimised the number of pulses to make sure I get a smear of between 100bp and 1Kb when I look at an aliquot on a gel following reversal of crosslinking)
I block samples with Rabbit IgG for 3-5 hours and treat with the antibody for the TF I am studying O/N.
The beads are agarose G. I do the RNase, reversal of crosslinks and Proteinase K steps, and do Phenol-Chloroform extraction to purify using Glycoden and carrier tRNA.
I have also done this exact procedure with the positive control H3K4me3. qPCR indicated an enrichment of a positive site by 40-50% so I assumed the ChIP had worked. However, the bioanalyser profile is the same as for my samples with a peak at 90bp and nothing bigger.
If anyone has seen this kind of thing before and has any ideas I'd be really grateful.
Thank you!
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