Has anyone tried using RE that creates blunt end (e.g. PvuII) for primary RE in 4C (Circularized Chromosome Conformation Capture) Assay?
My bait ("viewpoint") region has very limited RE choice...
Also, whoever has an experience with 4C, could you tell me how big the bait ("viewpoint") can be?
Thanks for all your helps and insights
Hi,
blunt end works, BUT, of course, blunt end is blunt end is blunt end !!!
BUT if I may be honest: why do you still use 4C? it has no good quality because of the PCR and you might not be able to see what you want to see one to all instead of in a local region everything with everything???
so the question is: what do you want to investigate?
if you want to know about a local region's interaction use T2C - much much better outcome! then you can really make a statement about what is interacting with what !
here the paper in print where you can see what you can do:
https://www.researchgate.net/publication/306107806_The_Detailed_3D_Multi-Loop_AggregateRosette_Chromatin_Architecture_and_Functional_Dynamic_Organization_of_the_Human_and_Mouse_Genomes_-_bioRxiv_Preprint_Version
if you want we can work together on this, then contact me by email: [email protected]
Article The Detailed 3D Multi-Loop Aggregate/Rosette Chromatin Archi...
ah and I forgot to mention: with 4C everything depends on your viewpoint, but are you sure about your viewpoint ???
Thanks for your insight Tobias
But essentially what I want to see is more simple. I have a specific region within genome (a promoter region) and want to look at the other possible regions that interact with this promoter region.
What other options do I have other than 3C-seq / 4C for this specific purpose?
Thanks!
the best is T2C - so not only to use one viewpoint, but an entire region as viewpoints - then you know exactly how the stuff acts locally and also with what else it interacts further away or even on other chromosomes - so that I do not say, because I invented T2C, but because I have seen so many people with suggestions who were easily fooled by 3C, 4C, or even HiC... - I guess if you read my paper you know what I mean...
Hi Donghyun,
I am not aware of any published 4C studies using blunt-end enzymes. From Hi-C studies it is known though, that ligation is considerably impaired. My advise would be to not use blunt end enzymes.
If you expect your interacting partners to be located at quite a distance (let's say over 100 kb), your viewpoint does not need to exactly overlap your promoter. When using a 4-cutter, you can design your viewpoint without problems up to 2-3 kb up- or downstream. For 6-cutters this is even more (5-10 kb). I hope this will help you to find a non-blunt cutter.
For your second question: there is no maximum size for your viewpoint. But do not make your viewpoint too small. For 4-cutters I would avoid going below 600-700 bp, for 6-cutters not below 1 kb. Also keep in mind that the distance between your first and second restriction enzyme at the viewpoint should be 250 bp minimum.
Good luck with the experiment,
Daan
well, what you say Daan is an educated guess, BUT it depends on the 3D structure where your viewpoint sits, i.e. you just cannot in general say what you just said. it is just depending on the very individual environment your viewpoint is located in, and it depends even on a view base pairs sometimes. although it might be wise not to go below 250bp between first and second restriction enzyme or have a minium viewpoint of 600-700 bp, the reality shows, that one just cannot really say. we have great interaction results with resolutions of 50 bp and bad ones for 500bp. therefore, I adviced to do T2C to really get a view where you know properly what is going on, all the rest is a question of believe etc. but does not sustainably clarify something... I would like to have this different, but the reality is the reality science wise...
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