Dear all,
I'm encountering an already mentioned issue (http://seqanswers.com/forums/showthread.php?t=11737).
I'm creating a new thread though, as my ChIP experiments are slightly different (targeting histone modifications) and as I've already tested a number of hypotheses regarding this issue.
As mentioned in the above thread, I encounter a DNA size distribution issue after the ChIP : before ChIP, average size of the DNA fragments is around 200bp (with ≈99% of the DNA below 600bp) ; after ChIP, average size of the DNA fragments is around 1000-2000bp (with variable proportion of the DNA below 600bp from one sample to another).
Figure 1 : DNA size aberration is systematic with all antibodies.
Figure 2 : DNA size aberration is systematic with all ChIP replicates.
MG##_Sonication : Sample processed right after sonication to check the DNA fragment distribution.
MG##_ChIP-Input : Unbound fraction of a mock ChIP (no antibody).
MG##_ChIP-'antibody' : Bound fraction with a given antibody.
I reverse-crosslink overnight at 65°C with strong shaking. I perform a 3h proteinase K treatment at 55°C. All samples are then silica column purified. The DNA size aberration is not a Bioanalyzer bias as I observe exactly the same thing on an agarose gel.
I first incriminated an insufficient reverse-crosslink, but additional reverse-crosslink in optimized conditions does not change the situation. I then incriminated presence of RNAs, but additional RNase treatment does not change the situation either.
I am now very sceptical as well as desperate regarding this situation. I would be very happy if you could share your experience on this issue.
I'm encountering an already mentioned issue (http://seqanswers.com/forums/showthread.php?t=11737).
I'm creating a new thread though, as my ChIP experiments are slightly different (targeting histone modifications) and as I've already tested a number of hypotheses regarding this issue.
As mentioned in the above thread, I encounter a DNA size distribution issue after the ChIP : before ChIP, average size of the DNA fragments is around 200bp (with ≈99% of the DNA below 600bp) ; after ChIP, average size of the DNA fragments is around 1000-2000bp (with variable proportion of the DNA below 600bp from one sample to another).
Figure 1 : DNA size aberration is systematic with all antibodies.
Figure 2 : DNA size aberration is systematic with all ChIP replicates.
MG##_Sonication : Sample processed right after sonication to check the DNA fragment distribution.
MG##_ChIP-Input : Unbound fraction of a mock ChIP (no antibody).
MG##_ChIP-'antibody' : Bound fraction with a given antibody.
I reverse-crosslink overnight at 65°C with strong shaking. I perform a 3h proteinase K treatment at 55°C. All samples are then silica column purified. The DNA size aberration is not a Bioanalyzer bias as I observe exactly the same thing on an agarose gel.
I first incriminated an insufficient reverse-crosslink, but additional reverse-crosslink in optimized conditions does not change the situation. I then incriminated presence of RNAs, but additional RNase treatment does not change the situation either.
I am now very sceptical as well as desperate regarding this situation. I would be very happy if you could share your experience on this issue.
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