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Thread | Thread Starter | Forum | Replies | Last Post |
ChIP seq - Different DNA size on agarosgel and Bioanalyzer | Leeenaaa | Sample Prep / Library Generation | 73 | 11-02-2014 08:45 AM |
question on DNA fragment size after shear the DNA (in Chip-seq) | kaixinsjtu | Sample Prep / Library Generation | 4 | 04-05-2012 04:36 AM |
Single stranded molecules run crazy slow on a DNA Agilent Chip | pmiguel | Sample Prep / Library Generation | 14 | 03-19-2012 11:13 AM |
ChIP seq - Different DNA size on agarosgel and Bioanalyzer | Leeenaaa | Introductions | 0 | 02-21-2011 01:23 AM |
Agilent Bioanalyzer 2100 detects no DNA | obischof | Sample Prep / Library Generation | 5 | 01-11-2011 08:15 AM |
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#1 |
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Location: United Kingdom Join Date: Apr 2011
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I ran one of my input DNA and all my ChIPd DNA on Agilent Bioanalyzer today.And I am getting different DNA sizes for my input and ChIP DNA samples.Input shows a distribution around 100-300bp while ChIp samples is distributed around 900-6000bp. I processed them together ,decrosslinked overnight and cleaned up using Qiagen PCR clean up kit. I diluted the input 360 X to get 100pg/microlitre and the ChIPd DNA samples 10X to 300-500pg/microlitre before running on the Bioanalyzer. I am using Agilent High Sensitivity kit which expired on 3rd May 2011. The ladder ran correctly. So I am not sure if this inconsistency is due to the kit.
What could be the reason for this difference? ![]() Input DNA ![]() ChIP DNA Last edited by rahulr1485; 06-01-2011 at 09:58 AM. |
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#2 |
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Location: Munich Join Date: Jan 2009
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theoretically, your IP target binds to long pieces of shearing-resistant chromatin which are heavily under represented in your input material.
however, i guess there are other problems. what is your IP target? what's your total yield of DNA in the IP as compared to the input amount? |
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#3 | |
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#4 |
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do you pre-clear your chromatin before IP? did you ever do a mock-IP + Bioanalyzer?
another possibility for your problem is unspecific DNA fragments attaching to your beads as you appear to get a hell of a lot of DNA in your IP. |
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#5 |
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Location: United Kingdom Join Date: Apr 2011
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Thank you for your reply. Yes, I did preclear it for 2 hours. I didn't run mock IP on bioanalyser, however, I checked it on nanodrop and it showed some negative value.
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#6 |
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Location: morges Join Date: Apr 2012
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Hello,
It is nice to participate to this forum! I experienced the same problem...does anybody know if we can shear again the DNA after IP or if it is not recommended? Thank you for your help!! |
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#7 |
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Location: Montpellier, France Join Date: Feb 2011
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I've just shared a very similar issue that I encountered on my histone modifications ChIPs.
There : http://seqanswers.com/forums/showthread.php?p=74004 For your information. |
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#8 |
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Location: UK Join Date: Aug 2011
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you could but then your peaks won't be centered around their original position
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#9 |
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Location: Montpellier, France Join Date: Feb 2011
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I would not do that, as the identity of this peak is not clear. If you hypothesize it corresponds to unspecific binding, there is not point to spend time and money trying to do anything with it.
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#10 |
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#11 |
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#12 |
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Hi rahulr1485 and MGineste,
What you are noticing is unfortunately an ideal example of epitope destruction during high energy chromatin shearing. What is occurring is that you are over-shearing your samples to the extent that you are destroying the epitope by ripping off the proteins from the DNA. High energy shearing also transfers quite a bit of heat to the sample during processing, contributing the reversing of the cross links during shearing. The gel/bioanalyzer results look great, but when you IP, you only pull down what has not been sheared extensively. A very good way to confirm that is to IP different shearing time points, and check the results on the Bioanalyzer. Since MGineste is working with histones, which are highly prevalent, a western of the sheared chromatin will not be a good confirmation. if rahulr1485 is working with transcription factors, then a western will illustrate the epitope destruction during the shearing process by running aliquots of the shearing time course on a SDS PAGE for western. How are you fixing your samples? and how are you shearing chromatin? |
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#13 | |
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Location: Montpellier, France Join Date: Feb 2011
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I may have a basic one : http://seqanswers.com/forums/showpos...10&postcount=2 This is why I'm insisting on the type of beads that have been used. |
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#14 |
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Hi MGineste,
Thank you for that link and data. While your theory might be plausible for people who do ChIP with carrier DNA, quite a few people including my lab, do not use carrier DNA or DNA blocked magnetic beads for obvious reasons. The "technical" explanation I gave, is based on work by us, and several other groups who have noted similar issues. Thank you Hamid |
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#15 | |
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Location: Montpellier, France Join Date: Feb 2011
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I keep your observation in mind. Thanks for the feedback. |
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#16 |
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Location: morges Join Date: Apr 2012
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Hi everybody,
We are using Dynabeads which are not blocked by any carrier DNA so I also think the reason may be due to something else...what I can tell you is that even if you get this strange pattern it does not mean that the sequencing will be bad... |
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#17 |
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Location: morges Join Date: Apr 2012
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Actually,
Could it be that what we see corresponds to ssDNA enriched by the IP...since I just red on this forum that ssDNA is running very slowly compare to dsDNA on the DNA chip? |
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#18 |
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Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008
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Post a picture of your gel/bioanalyzer chip. Yes, ssDNA can run larger than dsDNA on a bioanalyzer chip.
-- Phillip |
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#19 |
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Location: Montpellier, France Join Date: Feb 2011
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I reproducibly observed the same pattern. I may have an explanation for this, that I already posted there : http://seqanswers.com/forums/showthr...50&postcount=3
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#20 |
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Location: California Join Date: May 2013
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Can somebody please tell me what should be the ideal size of frangments after IP if samples are run at bioanalyser/ Agarose gel?
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