ChIP sonication:different smear in cross-linked and non-corsslinked samples - (May/22/2008 )

Hello everybody,

hope you can help me with this: I'm trying to get ChIP established in our new lab, and using the BIORUPTOR waterbath from Diagenode. I've tried different settings so far, but all give me too big smears (from 100 bp to the best 5 kb). However, if I try the same conditions under native, non-cross-linked conditions, I get perfect results for my smear. I've tried extending the reverse cross-linking time to 24h (always 65C), but to no avail. Is my different smear length due to unsufficient reverse-crosslinking, and if so, how can I improve that ( I cross-link at 1% FA, 10 min 37C, and quench with 0.125M glycine)?
Most grateful for any ideas (will reward with virtual cookies ;-))

Thanks a bunch!

trishitrish

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Best Choice ever , I use teh same one.

1. You need to find ideal crosslinking time and temp for your cells and cell numbers. I use 10 minutes, RT. If you overcrosslink, you will never get uniformly small chromatin frags.

2. Number of cells is very important. If you have more cells than the bioruptor can handle, it is never going to work.

Here is my conditions for a variety of adherent cells:

(in one tube):
lysate from about 7.0 million cells,
in 300-400 microliter of lysis buffer,
30 second pulse/30 second wait
20 minutes.

It may be different for your cells, but should not be very dramatically different unless you are using elephant cells.



Edit: I said .7 million cells earlier. That would be wrong!

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Hi celllcounter,

sending you a virtual chocolate chip cookie! Thanks, that's really helpful, I'll try your cross-linking/shearing protocol.

Just one question: do you use 7 or 0.7 mio cellls? Got a bit confused here. So far I'm using 1 mio cells/200 ul lysis buffer. Do you get your smear uniformly between 200 bp and 1 kb? OK, that's 2 questions now :-)
Thanks a bunch again,

trishitrish

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This analysis is right.
You may be over-crosslinked.
cells that over-crosslinked are hard to get good smear..

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Hi Trishitrish

What different conditions have you tried? To optimise my sonication (same sonicator) I have had to play around with cell conc, volume and time (always 30 sec on/off). That's for 10 mins cross-linking at RT with formaldehyde.
So I use a cell conc of 1mill cells/30ul, sonicate for 5 mins in a volume 500-1700ul and I get about 200-600bp consistently (using the 10ml diagenode tubes).

Good luck

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Hi all

I too am using the Bioruptor which I was told to leave on the high setting but my fragments are way too small even after 3 minutes of sonication. Can I take a poll of who uses the Bioruptor on Low, Medium and High ?!

Thanks to all the Bioruptor ChIP'ers out there.

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What's size is too small?

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Hi Clare,

I used the recommended Upstate conditions, 200 ul/10^6 cells, but apparently I have to concentrate that ... Thanks!

By the way, I lowered cross-linking time+formaldehyde concentration, and it is muych better, though not perfect yet...

Thank you all.

Let's sonicate the world ;-)

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200 - 100bp average size

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