Question: what are the control samples when using MACS to find ChiP-seq peaks?
0
gravatar for akivab2
5.1 years ago by
akivab2100
European Union
akivab2100 wrote:

Hi,

I am trying to find the peaks in arabidopsis ChiP-seq data. In the MACS tutorials they refer to control samples.

What are they supposed to contain?

Thanks

control chip-seq macs • 3.7k views
ADD COMMENTlink modified 4.9 years ago by Ann2.2k • written 5.1 years ago by akivab2100

or the input DNA, which is just genomic DNA not subjected to IP.

ADD REPLYlink written 5.1 years ago by Ming Tang2.5k
2
gravatar for Ann
4.9 years ago by
Ann2.2k
Concord NC USA
Ann2.2k wrote:

I think the macs2 tutorial is a little vague because there are many options for "control" sample.

For example, in my lab we are using input DNA as the control.

By that, I mean: DNA that you treated exactly the same way as the IP samples.

ChIP pipeline is:

  1. Cross-link protein to DNA (formaldehyde)
  2. Sonicate DNA (chop into fragments)
  3. Immunoprecipitate w/ antibody against transcription factor of interest
  4. Make libraries, sequence.

To get input, sequence the output from step #2. It controls for uneven sonication and cross-linking.

However, you can get more creative with this.

For example, let's say you have a line that expresses a mutant allele of your transcription factor - maybe it lacks a phosophorylation site you think is important for activation. Or maybe it has a mutatation that makes the phosphorylation site appear constitutively active. (A phosphomimic.)

You could put this line through steps 1 to 4 and then use the resulting sequences as the control in a macs2 analysis.

A cautionary note: When working with biology collaborators to design such experiments, make sure it will work for them by analyzing data sets from their species that have a similar design. Or be extra conservative and tell them to sequence both types of control.

ADD COMMENTlink written 4.9 years ago by Ann2.2k
1
gravatar for Devon Ryan
5.1 years ago by
Devon Ryan92k
Freiburg, Germany
Devon Ryan92k wrote:

Normally when you do ChIPseq you perform a control IP of some sort. The control IP would be the control sample.

ADD COMMENTlink written 5.1 years ago by Devon Ryan92k
1
gravatar for Chris Fields
5.1 years ago by
Chris Fields2.1k
University of Illinois Urbana-Champaign
Chris Fields2.1k wrote:

The control is the input DNA.  You can use a mock sample (IgG) but the recommendations from ENCODE and others is to use input, as it represents the true background and can help correct for bias.

ADD COMMENTlink written 5.1 years ago by Chris Fields2.1k
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