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Zohreh_fa's Content

There have been 5 items by Zohreh_fa (Search limited from 20-January 19)


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#178259 Bioanalyzer for ChIPed DNA-need help!

Posted by Zohreh_fa on 13 June 2016 - 12:43 PM in ChIP and Next Generation Sequencing

Dear All,
I have a question regarding bioanalyzer for ChIPed DNA.
When I do bioanalyzer for the input DNA, I get peaks at the proper range in bioanalyzer.
But, for the immunoprecipitated samples, I got no peaks at all or strange peaks. I even do not have the marker peaks!
I should mention that the concentration of ChIPed DNA was as low as 0.3 ng/µl (measured by Qubit HS kit) and I used 1 µl for bioanalyzer. So, I expected very low signals in bioanalyzer, but literally got nothing.
These samples were loaded in a same bioanalyzer chip used for some other library prepped samples and they all worked well, which shows that there was no problem in bioanalyzer chip or experiment.
Can anyone help me how to explain this?
I am totally confused and cannot figure out what I should do!
In the attached you can find a gel photo of the sonicated chromatin as well as two bioanalyzer graphs of two different ChIPed samples.

waiting for your replies...

Bioanalyzer for ChIPed DNA? - =spam edited=. Available from: https://www.research..._for_ChIPed_DNA [accessed Jun 13, 2016].

Attached Thumbnails

  • Ladder.png
  • ChIPed Sample1.png
  • ChIPed Sample12.png
  • gel photo after sonication.jpg



#178188 Acceptable Fold enrichment threshold fro ChIP?

Posted by Zohreh_fa on 25 May 2016 - 08:16 AM in ChIP and Next Generation Sequencing

Hi everyone,

I have recently performed some ChIP experiments and did qPCR for GAPDH promoter to check the specificity of the ChIP in RNA pol compared to IgG sample.

qPCR showed 2 fold enrichment of GAPDH promoter in RNA pol sample compared to IgG sample.

My specific question is; if 2 fold enrichment is acceptable or not? what is the threshold of that to consider a ChIP experiment specific enough to go for further steps of library prep and sequencing?

The specific TF which I am trying to do ChIP on, has no known target genes. So, there is no option for me to do qPCR on positive or negative binding regions of that gene to check the specificity.

I also did qPCR for GAPDH on the TF ChIPed DNA and the Ct value was near what I got for IgG sample. All these show that probably there is a specificity but the difference is quite low (only 2 fold). ( See the attached photo)

Can anyone help me in what is considered as the threshold for fold enrichment?

Cheers,

Zohreh

 




#177793 What do people use as sequencing control in ChIP-Seq

Posted by Zohreh_fa on 03 March 2016 - 02:30 PM in ChIP and Next Generation Sequencing

Hi everyone.
I am a new one in ChIP-seq and planning to do it for a poorly characterized TF.
We are going to do ChIP on transfected HEK293 cells with WT and 2 other mutant forms of our specific gene which makes three samples to be sequenced.
Regarding the negative control "Input DNA", the amount of samples for NGS would be 6 samples (1 sample+ its corresponding input DNA) which is quite expensive for us.
As I am searching to find a way to reduce the cost, I was wondering that if its possible to find the sequencing RAW files of control DNA for HEK cell somewhere in databases and use it as a control to normalize the analysis. Is it possible? 
Are there such kind of data available and if so, can we use them instead of input DNA?
As I understood the sonication is not a truly random process and we may have to sequence each samples's input DNA separately.Am I correct?
If so, can we sequence only one of the input DNAs (for example WT) as the control for all the three samples or we have to consider one seperate input DNA for each sample?
 
I am looking forward to hearing from you.



#177792 What do people use as sequencing control in ChIP-Seq

Posted by Zohreh_fa on 03 March 2016 - 02:29 PM in ChIP and Next Generation Sequencing

Hi everyone.
I am a new one in ChIP-seq and planning to do it for a poorly characterized TF.
We are going to do ChIP on transfected HEK293 cells with WT and 2 other mutant forms of our specific gene which makes three samples to be sequenced.
Regarding the negative control "Input DNA", the amount of samples for NGS would be 6 samples (1 sample+ its corresponding input DNA) which is quite expensive for us.
As I am searching to find a way to reduce the cost, I was wondering that if its possible to find the sequencing RAW files of control DNA for HEK cell somewhere in databases and use it as a control to normalize the analysis. Is it possible? 
Are there such kind of data available and if so, can we use them instead of input DNA?
As I understood the sonication is not a truly random process and we may have to sequence each samples's input DNA separately.Am I correct?
If so, can we sequence only one of the input DNAs (for example WT) as the control for all the three samples or we have to consider one seperate input DNA for each sample?
 
I am looking forward to hearing from you.



#176095 Chip Assay-Cell type?

Posted by Zohreh_fa on 27 July 2015 - 01:38 AM in ChIP and Next Generation Sequencing

Dear All,

 

I have a question regarding Chip - assay.

We want to use chip-assay to estimate target genes of a novel transcription factor which is assumed with a role in brain and nervous system.

 

My first question is, what kind of cell line I should start with.

Should I use a special neural cell line or any other kind of cell is proper for the work, for example lymphoblastoid cell lines?

 

Also, since this is the first time that I am going to do chip assay and there is no one in the lab with experience in the field,

I tried to find available kits for performing Chip in the lab.

For example, ChiP Kit (ab500) from abcam company or  Pierce Agarose ChIP Kit from Thermo Scientific.

Would you please kindly help me in this subject, if its better to use the commercially available kits or I should set it up in the lab manually?

 

Best Regards,

Zohreh

 




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