what are the control samples when using MACS to find ChiP-seq peaks?
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6.9 years ago
akivab2 ▴ 110

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

ChIP-Seq MACS control • 4.6k views
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or the input DNA, which is just genomic DNA not subjected to IP.

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6.7 years ago
Ann ★ 2.3k

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.

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6.9 years ago

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

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6.9 years ago
Chris Fields ★ 2.2k

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.

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