Sonicate for >60min: What did I do wrong?? - ChIP sonication disaster (Nov/18/2010 )
Hi Shan,
MNase is a non-specific endo-exonuclease cleaves most forms of nucleic acid, yet this cleavage is protected by binding to the nucleosome. So it basically cleaves DNA betwen the nucleosomes. Sorry about the image not appearing, it does on my pc. If you could see this you could see the ladder of difecernt sized bands coresponding to mono-, di-, tri-nucleosomes and so on. Maybe try searching for some gel images online, there must be some. You can get the desired fragment size, however I was informed by another post-doc in a neighbouring department that cleavage just using MNase alone can lead to a sequence bias in your ChIP-seq data and its better to use a mild digest to break into mainly larger stretches of nucleosomes and then sonicate down to the desired fragment size. Sadly as this was purely word of mouth I can offer up no solid conclusive evience for this. I do apologise.
As to the SDS issue I believe that SDS limits binding of the antibody to antigen and is often used in elution buffers to remove your precipitated complex from your beads. This shouldnt be a problem at low concentrations as you can easily dilute your samples to contain less. However I was having to go above 2% SDS and still sonicate for over 90minutes to recieve good shearing and this often still left some genomic DNA in the sample.
I think it is a case of having to try all available options and find which works best for your samples. I am working on meiotic cells and think that maybe the larger number of proteins binding the chromosomes together are creating the issue in my case, it is worth looking at all possibilities as it may not be an issue with your technique.
Ben
Thanks Ben!
Sorry to hear all that you had to go through with your meiotic samples... That made mine seem less complicated, since i did get desired fragment size although it's taking unbelievably long. And apologies are not necessary for providing brilliant information!
Now it sounds like soincation can be really complicated than deciding which power level and how long. I think I will give SDS a try first while waiting for the MNase to come in (6 min plus a round of sonication sounds really attractive!) So do you mind telling me which company you ordered it from and how long a round of sonication you meant?
Thanks!!
Shan
Ezilybored,
Nice Gel, im glad to see that you can MNase after cross-linking. So for your first lane, what volume was that undiluted MNase digestion performed in? Would you recommend using it for ChIP qPCR?
Shan,
no worries, its why im so glad to pass information along now and save other people a lot of time and hassle. The MNase is from Sigma. I am currently performing a 5 min sonication to get all material into solution, followed by a 1 minute digest with MNase (1U/ul) then following this up with 30 mins of sonication to get a 100-500bp spread. I could just go for the 6 minutes 10U/ul digest and get a similar result, however I am still concerened by this sequence bias issue I was warned about and willing to put in a little extra time to avoid it. Good luck with the SDS attempt in the meantime.
Chabraha,
I have now used my combination MNase/Sonication sheared chromatin to do 2 IPs and the results look very good by ChIP-PCR. We get enrichment at known binding sites and nothing at non-binding sites with sufficient yeild of DNA for checking by qPCR and subsequent sequencing also.
Ben
Chabraha,
sorry just realised you asked reaction volume too. It was a 500ul volume. I resuspended 10x7 cells in 1ml MNase buffer, split to 2 tubes of 500ul then sheared as above. When pooled afterwards this yeilds around 22-25ug sheared DNA from my cells.
Ben
Thanks Ben!
That was very clear and now I just need to see if my supervisor will let me have a try.
I don't really want to drag on this matter but I still can't quite shake my head to understand the sequence bias thing. Could you give me a reference to read about the pros and cons of using MNase? I will really appreciate it!
Thank you!!
Shan