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Old 07-03-2014, 01:14 AM   #1
HSV-1
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Angry ChIP qPCR vs ChIP-seq

Hi ,

We have performed Chromatin immunoprecipitation experiments.
Our goal is to find out the genome-wide bindings of a transcription factor so we sent our ChIP-qPCR validated sample to do the illumina sequencing.

Finally we were frustrated by the ChIP-seq results which turn out to be random reads mapped to genome.

What's wrong with the ChIP-seq? Is it common or possible that ChIP-qPCR doesn't fit ChIP-seq?
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Old 07-03-2014, 06:10 PM   #2
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How many sites did you test by qPCR? Were the qPCR sites highly enriched? Did you test both positive and negative sites by qPCR? We've had a few times where the qPCR suggested the ChIP worked but didn't work well and then the chipseq didn't really work at all, because the qPCR is more sensitive it's more forgiving to a ChIP that didn't work so well. If you are able to detect robust enrichment at several sites by qPCR and also have clean negative sites I would think your DNA is good enough for chipseq. Was the DNA tested on a Bioanalyzer and did it look ok? Same with the library? Even though your reads seemed to map randomly did you still get a high percent alignment? What was the duplicate level like?

Last edited by biocomputer; 07-03-2014 at 06:14 PM.
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Old 07-04-2014, 11:15 PM   #3
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Quote:
Originally Posted by biocomputer View Post
How many sites did you test by qPCR? Were the qPCR sites highly enriched? Did you test both positive and negative sites by qPCR? We've had a few times where the qPCR suggested the ChIP worked but didn't work well and then the chipseq didn't really work at all, because the qPCR is more sensitive it's more forgiving to a ChIP that didn't work so well. If you are able to detect robust enrichment at several sites by qPCR and also have clean negative sites I would think your DNA is good enough for chipseq. Was the DNA tested on a Bioanalyzer and did it look ok? Same with the library? Even though your reads seemed to map randomly did you still get a high percent alignment? What was the duplicate level like?
Thanks ! I will be careful with the coming experiments.

More questions:
Once ChIP DNA is purified how long it could be stored at -80 (a few weeks) or Liquid nitrogen(forever)?

How did you purify your ChIPed DNA ? by column or by precipitation with glycogen?


Thanks again!
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Old 07-11-2014, 02:56 AM   #4
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We purify with the Qiagen PCR Purification kit and store purified DNA in the kit's elution buffer at -20 for at least 6-12 months without any problems, it's probably fine for longer than that.
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