Hi Everyone,
I am trying to optimize ChIP for sorted CD8 T cell obtained from mouse splenocytes(primary cells). I am doing the ChIP with a kit from Cell Signaling Technologies[SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003]. I kept 5 million sorted naive CD8 T cell for activation and after 3 days I got 8.5 million activated CD8 T cell. I did the entire process following the protocol provided in the kit word by word. it was mentioned in the provided protocol to use 0.5 μl Micrococcal nuclease for 20 mins and sonicate with three 20 sec pulses on bath sonicator with 30 sec gap between pulses which yeilded me 81ng/ μl of DNA after purification. but when I ran the sample on 1% DNA gel I found that my DNA sample has been overdigestedsince it was mentioned that DNA fragment size need to be in between 150bp to 900 bp for ChIP.
I looked for papers regarding this thing but found none that used the kit for primary cells.
Can anyone give me any suggestion that I can use for my next set of experiments?
should I reduce the Micrococcal nuclease digestion time or concentration? or should I use less pulses instead?
I am attaching the photo of my gel and the document portions in this question.
Likely you do not need to do both MNase digestion and sonication. I would personally stick to one method. In the past I have used sonication and had successful ChIPs. You may prefer MNase digestion as it is enzymatic and may cause less damage to the ends of the DNA, but personally I would not combine them.
Actually It was recommended for HeLa in the protocol that came with the kit and i followed the exact thing the first time. I'll keep your recommendation in mind and try it in the next set of experiments
Hi Anwesha. As Joshua Beytebiere already mentioned, using both sonication and MNase digestion on one sample is rather unusual. Nevertheless you are correct with the assumption that your chromatin might be overdigested if you want to go for 150-900bp, assuming the lowest marker band is 100bp. Both MNase and sonication treatment can be altered to achieve this as you already pointed out, but which to choose propably comes down to what you are trying to achieve. Sonication is considered more "rough", but less biased while MNase digest is more "gentle" but slightly more biased. Additionally, what protein is your target in this ChIP? This might be a key factor on which method you should focus on.
Sadly, I don't have a lot of expierence on sonication so far, but from what I have read Joshua Beytebiere is very experienced in this field and should be able to pinpoint what exactly you should tweak. As for MNase digest, many factors will determine your final outcome (time, temperature, concentration of both MNase and chromatin, the MNase you are using, Ca2+ availability, ...). Personally, I would recommend to stick to 37°C, 10min (optimal with shaking at approx. 500rpm) and do a titration of your MNase with aliqouts of the same starting material and varying MNase concentrations. Afterwards, do the general RNase, Proteinase K and purification steps and analyze by agarose gel. If you want to go for smaller fragments (say 300bp or so) I would also recommend to use a 1.5% agarose gel for a better resolution of smaller fragments.
I hope this might help you.
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