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hi all,
we are planning on doing a ChIP-Seq experiment trying to identify PolIII binding sites in yeast and mouse.
As I am not really sure of the binding affinity of PolII I would like to ask for your opinions.
First a general question. I know PolII has a mixed binding pattern (narrow and broad BS). But I am not sure about Pol3. Does it also have a mixed behavior or is it more similar to the point-source factors (TF)?
I would also like to ask for your opinion as to how deep to sequence and how many replicate are necessary to hope for good and valid results.
I know the Encode consortium recommend 20M mapped reads for human for point-source Binding events, but as we are talking about a possible mixed behavior and also partially doing it in yeast, I was wondering how many reads are necessary for these two organisms?
Another question I have just thought of is about the control. Which control samples are better - IgG controls with "mock" antibody, or the input Samples from similar cells, but without the antibody?
Which of the two controls are better for the this kind of analysis? As fat as I know, there are no tools, who can work with both of them.
Thanks in advance for any help
Assa
we are planning on doing a ChIP-Seq experiment trying to identify PolIII binding sites in yeast and mouse.
As I am not really sure of the binding affinity of PolII I would like to ask for your opinions.
First a general question. I know PolII has a mixed binding pattern (narrow and broad BS). But I am not sure about Pol3. Does it also have a mixed behavior or is it more similar to the point-source factors (TF)?
I would also like to ask for your opinion as to how deep to sequence and how many replicate are necessary to hope for good and valid results.
I know the Encode consortium recommend 20M mapped reads for human for point-source Binding events, but as we are talking about a possible mixed behavior and also partially doing it in yeast, I was wondering how many reads are necessary for these two organisms?
Another question I have just thought of is about the control. Which control samples are better - IgG controls with "mock" antibody, or the input Samples from similar cells, but without the antibody?
Which of the two controls are better for the this kind of analysis? As fat as I know, there are no tools, who can work with both of them.
Thanks in advance for any help
Assa