hello everyone, I am new in chip-seq. Recently I want to try arabidopsis thaliana chip-seq by following this protolcol: Article Profiling histone modification patterns in plants using geno...
I have applied 2 g of A. thaliana seedling to follow the steps describe above, and I used Qsonica Q125 sonicator instead of Sanyo Soniprep 150. However, I failed to shear DNA into
Did you add RNase followed by proteinase-K before running the gels? For me, the first picture looks like RNA contaminant rather than sheared genomic DNA. To test sonication efficiency, a plasmid is not a good indicator. You need to use isolated genomic DNA (clean without RNA contamination), better from cross-linked samples.
Hendry Susila thanks for the answer! I know proteins bound to DNA will affect how the DNA runs on a gel, and I forgot to mention that first lane of the gel is before sonication, and the second lane is after 10 minutes of sonication, and third lane is 100bp marker.
I have cross-link DNA and protein with formaldehyde (with vacuum infiltration), and after nuclei isolation and sonication, I directly run the gel before immunoprecipitation and reverse cross-linking to test the sonication efficiency. As mention in the protocol above, I should see a difference between a DNA smear from the sample that was sonicated and the one that was not. However, it seems like the "smear" on my gel is RNA contamination.
Where is the problem? is the black arrow indicate the uninterrupted genomic DNA?
The sonication time I read in most of the papers said that total sonication time of 2 minute is sufficient, and I'm pretty sure I have directly inserted the sonication probe in my sample.
By the way, most of the papers will not treat RNase but proteinase K after reverse cross-linking.
Looking forward to your reply.
Yes, the smear is likely RNA. The black arrow seems like gDNA, but it's too close to the well. Did you purify the DNA before loading it onto the gel, either by column or the phenol:chloroform method?
Sonication time really depends on the machine; in my case, it's 30 seconds on, 90 seconds off for 10-15 minutes. RNase is crucial to remove RNA contaminants, ensuring what you observed is sheared DNA.
I wrote a ChIP-qPCR protocol for Arabidopsis, which you can find at https://pubmed.ncbi.nlm.nih.gov/33421000/.
I hope it helps. Good luck.
Hendry Susila Hi there, your protocol helps me a lot!
But I have little question:
As suggest in your protocol to test the sonication efficiency, I directly did reverse cross-linking after sonication, then added proteinase K and RNase, and purified DNA with kit.
The gel image below is I tried different sonication condition (first lane is unsonicated, 2-4 is different sonication condition, 5th lane is 100bp DNA marker). It seems the sonication efficiency is visible after DNA purification
Do you think any of these condition is suitable for doing ChIP? If any of these conditions are suitable, I will proceed with the immunoprecipitation next time. If not, I will try another sonication condition.
Hi, I'm unsure about the DNA marker size, but a smear ranging from 100-1000bp is suitable for ChIP. I guess lanes 2 and 3 are good enough. Good luck!
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