Hi everyone,
In order to do CHIP experiment, I used some lysis buffer and micrococcus followed by sonication to digest chromatin of the cells. I did all the process of cross-lincking, quenching, and cell lysis and chromatin digestion and then DNA purification and then I loaded the DNA in gel agarose but I see a lot of smear in longer fragments. Can someone guide me how can I know that these smear belong to the un-lysis cells or un-fragmented chromatin. how can I be sure that they are not chromatin. I uploaded the gel image and these huge smear in the large part of the gel is visible.
Hello,
From my experience this is a fairly normal sonication pattern maybe a little bit of high molecular weight fragments but not too much. With sonication you can pretty much always expect some kind of smear. Have you tested different amounts of sonication? It is likely fragmented chromatin however it should not cause your experiment issues. If you are doing ChIP-seq usually during the library process you will go through a size selection and the larger fragments will be removed so I would not worry about this unless you have the majority of your chromatin as high molecular weight. I would personally shoot for > 80% to be between 100-1000bp. What are your sonication conditions? Is there a reason you are using an MNase digestion and sonication? I personally would simply just sonicate the chromatin.
Hi,
I agree with Joshua Beytebiere . just sonication should be enough. then many parameters can influence chromatin prep. do you isolate nuclei? fixation protocol? cell concentration?.....
In general the best is to perform a time course with your sonicator to find the best sonication conditions. then stick to that.
Here is a guide to help you optimize
https://www.diagenode.com/en/pages/chromatin-prep-easyshear-kit-guide
Hi,
It's totally normal as mention above. Lots of people try to remove the smear by adding sonication to increase the amount of chromatin recovery. As I can see you have a nucleosomal pattern which means than you are sharing preferentially nucleosomal DNA. Regarding the type of experiment you could have trouble with recovering some sequences (especially for Transcription Factors). This ladder is due to a too-high sonication. If you need to increase the small size and keeping most of the sequences such as open chromatin, I suggest to use the nexon-CHIP protocol. It had a pre-sonication step to prepare nuclei before the sonication which is useful because most of the time lysis step do not lyse cells due to fixation.
https://pubmed.ncbi.nlm.nih.gov/26704968/
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