I'm trying to do a DNA/RNA immunoprecipitation (DRIP) using the S9.6 antibody and am getting no DNA pull down. I isolate DNA from cultured cells using the Qiagen DNeasy Blood & Tissue Kit and then proceed with the DRIP as outlined by either -
1. Experimental design # 13 in http://genome.cshlp.org.ezp-prod1.hul.harvard.edu/content/27/6/1063.long
or
2. https://link-springer-com.ezp-prod1.hul.harvard.edu/protocol/10.1007%2F978-1-4939-6716-2_13
Trying both protocols, I have failed to pull down any DNA. Do you think the Qiagen DNeasy Blood & Tissue Kit could be destroying my R-loops? Does anyone have a protocol that works well?
Thanks!
I can't access your protocols, but I would think that given the RNase present in the DNeasy kits as well as its efficiency in removing protein from DNA would result in the loss of your R-loops. Can you do a nuclear isolation (like that done for ChIP experiments) and move forward from there? I'm very interested in whatever you find out that works.
I've used this kit to isolate nucleic acids, and I was able to see a signal using the s9.6 (R loop recognizing) antibody on a slotblot, so I think that isolation using this kit does not lead to loss of R loops. However, I indeed skipped the RNAse step, as William H. Broach suggested before.
Could you perhaps upload a pdf or word of the two links that you mentioned? We can not access the sites due to username and password restriction.
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