I'm working with the recently published Nature protocols 3C-Seq protocol on fibroblasts. After lysing the cells with the hypotonic buffer (with or without additional dounce step), I re-suspend the nuclear pellet with NEB buffer 3 (1.2X). When I add SDS (final 0.3%) the nuclei aggregate and form big clumps. How can I avoid this?
Longer lysis can help the digest quality, I've tried slightly higher detergent concentrations in the lysis buffer as well, both help with difficult cell types, including fibroblasts. With regards to the clumping, treat the nuclei as gently as possible after lysing. 200g spins (beware of loose pellets), VERY gentle resuspension, buy non-stick tubes and rotate the samples end over end instead of shaking them. These can all help you sufficiently digest the chromatin and avoid clumping.
My impression is that this clumping is caused by poor lysis. Matthew's suggestion are therefore a very good start. Additionally, it never hurts to use a cell strainer, even on cultured cells.
If you still get clumping afterwards, you can incubate your cells 10 minutes at 65 degrees after adding the SDS (like done in this HiC ptotocol: http://www.sciencedirect.com/science/article/pii/S1046202312001168). High temperatures will revert cross-links though, so try to avoid this step when possible and definitely don't incubate longer than 10 minutes.
Am facing the same issue, but in my case am getting clumps after adding TritonX. Mr.Izhak it would be nice if you could share with us as to how you solved this issue.
I am also having problems with cells clumping after the addition of SDS. I have been using a few pulses with a sonicator at the lowest power (5 cycles with power output at 3% with a 30% duty cycle) to break them apart. Microscopy shows that I still have a high number of intact cells following this treatment.
Is this really a question of lysing or just permeablization/extraction? I was under the impression that the lysing step removes the cytoplasm as much as possible to give you intact nuclei, but if there is sufficient extraction of and the enzyme can get to the nuclei this should not be a problem. Is my logic flawed on this?
I have tried this protocol with K562 and other no fibroblast cells and get good results but normal diploid fibroblasts seem to be the most problematic.
I am also working in a chromosome conformation capture protocol (4c-seq). I have a doubt in the ligation steps about the usage of PEG, is it useful/appropriate using this reagent? If so, which final concentration is it recommendable using?
Thank you very much for your advice!
Detergent-free method for nuclei isolation is available at:
https://inventbiotech.com/products/minute-detergent-free-nuclei-isolation-kit?_pos=1&_sid=3e52cfd28&_ss=r
Vandhana Chez sorry for the late question. Did you solve your problem ? I also observe large blobby structure (nuclei aggregating) after addition of Triton just before ligation. Is it due to harsh handling of the nuclei ? or too high concentration of Triton ?
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