shearing chromatin with enzymes - (Aug/17/2005 )
I just read a ChIP method where the DNA was sheared by enzymatic digestion, BEFORE the the nuclei were lysed. I am a little confused by this - can someone tell me if the enzyme is able to pass through the nuclear pore to cut the chromatin? Or am I just totally missunderstanding something?!?!
Any help would be appreciated !!!
haven't heard of this before, can you tell us where you found the protocol? is it online????
The protocol is from a confidential report - hence I can't actually ask the authors!
I have always sheared my chromtin by sonicating, but wanted to use the same antibody for my ChIP that this group had used so thought I'd follow their optimised protocol...
I have the same conern about this. I am working on tissue ChIP with the Activemotif enzymatic shearing kit. In this protocol the enzyme cocktail is added directly into nuclei suspension. Actually, the shearing of chromatin works well, but the problem is that I can not have the sheared chromatin in the supernatant. I guess the nuclei membrane is not broken. Does somebody have any idea about this issue?
In most of current chip protocols out there, cells are washed with PBS then lysis buffer, which spin with low speed. So the cells are loosen up for enzymatic digestion--MNase.