2018 - Large fragments observed as well
Hi, guys.
The same here. I am preparing ChIP-Seq samples for a "co-factor". There is only 1 paper reporting it binds to DNA at all. In a different cell line
My IP on well sheared chromatin yields much more >1 kb fragments than the smaller ones. When DNA yield in total is about 10-40 ng/IP, the DNA yield for <=400 bp is in the range of pg!
Two totally different protocols are already tried, including enzymatic chromatin digestion and sonication. No help.
Thoughts:
- might try to super scale up to get more total DNA (to increase the yield of <400 bp fragments);
- there shall be no DNA >400 kb fragments before IP. I may try to oversonicate/overdigest chromatin before IP;
- I want to try DNA beads (Select-a-Size DNA MagBeads from Zymoresearch) on my lysate to remove >400 bp fragments before IP ... not sure how it would work out as there is SDS, proteins, NP40 etc etc in the cell lysate;
- digest/sonicate DNA after IP
... If the pull down of larger fragments is specific, then why not? On the other hand, this shall bring irrelevant DNA fragments just situated in between of the "co-factor" binding sites;
- finally, they guys who published that only one paper on this co-factor, prepared the library on what they had and manually excised <400 bp fragments from from the gel. May be this is the right answer...
Any comments will be very much appreciated
Hi, guys.
The same here. I am preparing ChIP-Seq samples for a "co-factor". There is only 1 paper reporting it binds to DNA at all. In a different cell line
My IP on well sheared chromatin yields much more >1 kb fragments than the smaller ones. When DNA yield in total is about 10-40 ng/IP, the DNA yield for <=400 bp is in the range of pg!
Two totally different protocols are already tried, including enzymatic chromatin digestion and sonication. No help.Thoughts:
- might try to super scale up to get more total DNA (to increase the yield of <400 bp fragments);
- there shall be no DNA >400 kb fragments before IP. I may try to oversonicate/overdigest chromatin before IP;
- I want to try DNA beads (Select-a-Size DNA MagBeads from Zymoresearch) on my lysate to remove >400 bp fragments before IP ... not sure how it would work out as there is SDS, proteins, NP40 etc etc in the cell lysate;
- digest/sonicate DNA after IP
... If the pull down of larger fragments is specific, then why not? On the other hand, this shall bring irrelevant DNA fragments just situated in between of the "co-factor" binding sites;- finally, they guys who published that only one paper on this co-factor, prepared the library on what they had and manually excised <400 bp fragments from from the gel. May be this is the right answer...
Any comments will be very much appreciated
Comment