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We've done several ChIP experiments that we want to sequence. We sent our samples to the sequencing facility and were told that they had bad Bioanalyzer results. We then used our own Bioanalyzer and retested some of the leftover samples and also prepared new samples and tested them. After ChIP the DNA is purified using a Qiagen column and eluted in the Qiagen elution buffer (10 mM Tris·Cl, pH 8.5). The below analysis is on the purified ChIP DNA not prepared libraries.
Here are some examples: http://i.imgur.com/rDXjTJi.png
1. Old input used as a control and I think it's the only one that looks good
2+3. New inputs, they have 2 peaks and one has a dip at ~300bp.
4+5. IP samples, both have low concentration and one has a dip at ~300bp.
Here are some other samples: http://i.imgur.com/BbC8Fkr.png
These are two samples tested at two different facilities at two different times. They all seem to show low concentration but only one facility shows the dip at ~300bp. I've asked the people who run both Bioanalyzers and neither know what this dip is, though one said that he had seen it a few times before with other peoples' samples.
All samples tested by realtime PCR at sites of interest amplify as expected. We have sequenced other ChIP samples in the past but at different facilities and were never told of any problems with our DNA, but I don't know if that's because we really had no problems or because they didn't run the samples on a Bioanalyzer. Is it standard practice to test the ChIP'd DNA on a Bioanalyzer before library prep or anything else is done with it? The sequencing results we got in the past were generally ok though a few times we had high duplication rates.
So what could be going on here? Why do some of the inputs have two peaks, do some of the samples just have really low concentrations, what is the dip at 300bp and why would it only show up when a sample is tested at one place but not another? Is it worth it to try make libraries and sequence these?
Here are some examples: http://i.imgur.com/rDXjTJi.png
1. Old input used as a control and I think it's the only one that looks good
2+3. New inputs, they have 2 peaks and one has a dip at ~300bp.
4+5. IP samples, both have low concentration and one has a dip at ~300bp.
Here are some other samples: http://i.imgur.com/BbC8Fkr.png
These are two samples tested at two different facilities at two different times. They all seem to show low concentration but only one facility shows the dip at ~300bp. I've asked the people who run both Bioanalyzers and neither know what this dip is, though one said that he had seen it a few times before with other peoples' samples.
All samples tested by realtime PCR at sites of interest amplify as expected. We have sequenced other ChIP samples in the past but at different facilities and were never told of any problems with our DNA, but I don't know if that's because we really had no problems or because they didn't run the samples on a Bioanalyzer. Is it standard practice to test the ChIP'd DNA on a Bioanalyzer before library prep or anything else is done with it? The sequencing results we got in the past were generally ok though a few times we had high duplication rates.
So what could be going on here? Why do some of the inputs have two peaks, do some of the samples just have really low concentrations, what is the dip at 300bp and why would it only show up when a sample is tested at one place but not another? Is it worth it to try make libraries and sequence these?