QC: nucleosomal-length fragments

[Minglu1998]

Dear Ye,

Thank you for this very well written tutorial!

I have a very simple question about the QC step - nucleosome length that you mentioned in section 3.4:

"CUT&Tag reactions targeting a histone modification predominantly results in fragments that are nucleosomal lengths (~180 bp)"

According to wikipedia , nucleosome core particle consists of 146bp DNA. And the linker DNA is about 80bp.

My question thus is, when we check the QC plot, why are we looking for peak at about 180bp? What could be the range of acceptable length?

Thank you very much!

Bests,
Minglu

[yezhengSTAT]

Hello Minglu,
Thanks for bringing it up for discussion. Originally, I want to emphasize the periodic pattern in terms of fragment length as an indicator of a successful experiment. I also consulted with my colleagues regarding the 180bp fragment length pattern. He provided some potential explanation of the longer DNA sequences:

"As far as why the mononucleosome distribution has peaked at 180 instead of 147, I am not totally sure, but I guess it is due to (1) the Tn5 is tethered but will not necessarily insert right at the edge of the nucleosome, and (2) there may be some steric hindrance of the Tn5 itself against the nucleosome. A range of 160-200 bp for the mononucleosome peak is probably fine, although a pronounced ladder is typically indicative of a high-quality library."

Hope it helps.

Thanks,
Ye