What is ChROseq and how does it work

At the moment of writing this post, I’m sitting on the plane flying to Cornell. There I will learn a beautiful technique called Chromatin Run-On and Sequencing, or ChRO-seq.

Thirteen hours of travel is getting to the point of breaking my soul – I have finished most of work I can comfortablely do with one screen on a samll laptop. So I decided to write down what I know so far about ChRO-seq in preparation for my upcoming training tomorrow. I will keep updating this post as I learn more about this technique.

What is ChROseq

ChRO-seq is a variant of Run-On sequencing developed by Danko lab at Cornell (Chu et. al., 2018). In essence, it profiles nascent RNA by capturing RNA polII and DNA interaction. Below is a diagram from the authors depicting the major steps of a ChRO-seq experiment:

Summary of ChROseq methods

As shown above, insoluble chromatin is first isolated from tissues or cell culture. Isolated chromatin then gets re-suspended through sonication. Run-on reaction is done by incubating RNA polII and DNA complex with biotinylated rNTP. A complementary biotinylated NTP gets added on to the attached nascent RNA and prevents any further elongation.

Once run-on reaction is completed, 3 rounds of streptavidin bead purification are performed to purify extended nascent RNA, add 3’ adapter, remove 5’cap and add 5’ adapter. After adapters are added to both ends, a cDNA library is constructed from the nascent RNAs and a single end illumina sequencing is performed from 3’end.

What does ChRO-seq tell me

Like any run-on sequencing techique, ChRO-seq precisely captures nascent RNA when compared to general RNAseq. This means that:

  1. ChRO-seq gives a more accurate quantification of DNA transcription, as opposed to RNAseq, which describes a result of DNA transcription and RNA degradation.
  2. The fact that ChROseq is not noised by RNA degradation also means that it is able to capture many non-coding RNAs that have a short life span in cells and thus difficult for RNAseq to capture.
  3. ChRO-seq provides a snap shot of transcription activity. As revealed by other run-on sequencing, it can identify RNA polII pausing sites.
  4. Like other run-on sequencing, ChRO-seq can also be used to identify different types of transcriptions, as observed by Core et. al., 2008.

What does ChRO-seq not tell me

Because ChRO-seq captures nascent RNA, naturally, it does not contain any post-transcriptional level information like splicing, polyadenylation, and other RNA or protein modifications.

RNAseq ChRO-seq PRO-seq GRO-seq
RNA captured mature RNA Nascent RNA Nascent RNA Nascent RNA
Quantify transcription Yes(with degradation) Yes Yes Yes
Single base resolution Yes Yes Yes No
Material type RNA Insoluble chromatin Nuclei Nuclei
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Sichong Peng
PhD student

I study equine genetics/genomics at UC Davis Veterinary school. My primary interest is functional annotation of non-model organisms and its applications.

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