I'm looking for a kit to do ChIP-seq from low starting matherial (mice embryonic tissue). Diagenode True Micro Chip seems to be the best option, but I need to know the amount of chromatin needed per IP. The protocol is optimized for cells, starting from 10.000, but in my case the number of cells is difficult to measure whereas I can measure DNA concentration easily after sonication.
Thank you in advance
hello Irma!
I'm actually performing ChIP-seq analyis using exactly that kit from Diagenode on FACS sorted cells (Hystones modifications) and it works pretty well. I manage to perform comfortably 3/4 antibodies together sharing the chromatin from 150-250K cells. Trascription factors with this kit are more tricky to analyse.
Indeed, you can not check the sonication if you are short in samples quantity: what you could do is to perform some sonication tests and use everything to check the sonication efficiency (the parameters are really sonicator dependent) than apply them to your samples and perform the ChIP blindly. Normally I perform the test on the negative fraction of the cells, check it on gel and apply the same to my samples before ChIPing. In any case, I pool the surnatants after the IP and use it to have an actul confirmation of the chromatin size I have in my ChIped samples.
I have estimated someting like 0.5-1 µg of chromatin/million of cells (avarage) and the minimum amount to run on a gel around 1-200ng (small wells).
I guess you could do ChIP with homemade reagents, but this kit works very well!
Hi Irma,
The theoretical amount corresponding to 10.000 cells is 60 ng as one human cell contains around 6 pg of DNA. I hope that it will help you. If you have any specific questions you can contact me directly at [email protected]
Best regards,
Hello Filippo and Irina,
sorry for the late reply and thanks for your answers.
I also got another reply to my question from Diagenode technical service saying that although they have not performed an extensive validation of the kit on tissue samples so far, they run successfull ChIP experiments using the True MicroChIP kit on 200 ng of chromatin per IP.
So, I guess the minimum is between 60 and 200 ng (depending on ab, type of sample, etc).
Many thanks,
irma