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I had a few questions raised by the below diagram in relation to how ChIP-seq reads look like (source). I would be very grateful for your insight on them!

From the below diagram, it seems that, in a ChIP-seq experiment, we get information about the binding site in relation to both the Watson and Crick strands. That is, from our experiment, we get reads complimentary to both strands.

(Q1) I suppose this is because proteins bind to both strands of DNA, so that in our ChIP-seq experiment, we ultimately sequence reads from both the Watson and the Crick strands? Is that true?

Also, my impression from the MACS paper was that "ChIP-Seq tags represent only the ends of the ChIP fragments, instead of precise protein-DNA binding site" (Background).

(Q2) Therefore, shouldn't it be that we get reads mapping to either side of the binding site on BOTH strands? That is, we get reads more towards the 3' end of the Watson strand, and reads more towards the 5' end of the Watson strand, and similarly for the Crick strand.

(Q3) The diagram below shows we only get reads more towards the 5' end of the strands. Why is that?

(Q4) Finally, I know that MACs shifts reads by some bases towards the 3' end for each strand, in order to interpret the precise binding site location. It seems to assume what I am asking about in (Q3). However, if the answer to my (Q2) is "yes, we do get 4 such clusters of reads", then why doesn't MACs account for that?

enter image description here

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  • $\begingroup$ Some transcription factors bind to a DNA promoter sequence near the transcription start site and help form the transcription initiation complex. Do you mean transcription factors (TFs) in your Q1? $\endgroup$ Oct 26, 2022 at 3:31

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"ChIP-Seq tags represent only the ends of the ChIP fragments, instead of precise protein-DNA binding site" (Background).

(Q2) Therefore, shouldn't it be that we get reads mapping to either side of the binding site on BOTH strands? That is, we get reads more towards the 3' end of the Watson strand, and reads more towards the 5' end of the Watson strand, and similarly for the Crick strand.

Sequencing-by-synthesis (the approach used by Illumina which is the most common tech for ChIP-seq) always proceeds 5'->3' because of the way DNA is synthesized. So you read off only the 5' ends of the fragment, but not the 3' ends.

(More precisely: the 3' ends are the template, so the 5' is what you see in the read.)

Thus you only expect (start positions of) the reads to be at the 5' of each strand, as shown in the figure. So we only get 2 "clusters of reads", as implied by the figure and the MACS workflow, which only shifts 3' as you say.

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