Hi everyone, I am performing ChIP-seq on a transcription co-factor, for that I had to first crosslink protein-protein with EGS (1.5 mM) for 25 min and then I added 1% formaldehyde for 10 min, finally I quenched with glycine (125mM) for 5 min. I followed the iDeal ChIP-seq kit for transcription factors and for the sonication I used the Covaris machine. After checking fragment sizes I proceeded with decross-linking, IP and elution. I run the Qubit and I got for the input 4.8 ng/uL and for my IP 0.09 ng/uL, then before starting library preparation I run the Bioanalyzer for the input and I had big DNA fragments as you can see in the picture. I don't want to do bioanalyzer for the IP because I have very low starting material and I won't get a good quantification before library preparation. Do you think I should try to do a second round of sonication (on part of the input first and then once optimised, also on the IP sample)? Could you tell me if there is some troubleshooting I can do? Unfortunately it is very difficult to sonicate after so long cross-linking, but for transcription co-factor I can't do it otherwise.
THANK YOU in advance
This is a tough call - to clarify, you checked the fragment sizes before decrosslinking and doing the IP, and they looked okay? But then after the IP you checked the input again and they were too big? That's mysterious.
I want to tell you that it should be fine to optimize your sonication on the input and then do the IP sample, but given that the most labour-intensive and costly part of ChIP-seq is the library prep and the actual DNA sequencing run, in your position I would start all over again and be extra sure that the fragment sizes are right BEFORE the IP. I'm not sure what your exact experimental conditions entail, but growing and treating cells is cheap compared to sequencing a bad sample. I just wouldn't want to risk it. Good luck.
I am confused with two steps:
1) the DNA fragment size was checked before decrosslingking, and I want to know how did you shear the chromatin ,extracted the DNA and checked the size.
2) you said you decrosslinked the chromatin and did the IP, but in my experience, the decrosslinking was performed after IP.
In my opinion, optimizing the shearing conditions and ensuring a suitable DNA fragment size before IP may be useful.
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