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Old 08-11-2014, 02:42 AM   #1
friedrim
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Default ChIPSeq after FACS

Dear all,
I need to perform ChIPSeq on a subpopulation of my neuroblastoma cell line. However, I am worried that the FACS will compromise the integrity of DNA protein interactions. I am going to look at chromatin marks as well as transcription factor myc.
Does anyone have experience with this topic? What nozzle size and pressure do you use for cell sorting? In our facility, we have an Aria I machine, and I would like to use a 85M nozzle.
Did anyone try and do the crosslinking before vs after the cell sorting?

Many thanks for any comments.
Mona
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Old 08-11-2014, 02:51 AM   #2
dpryan
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If you don't get any good responses then you might contact Stephan Bonn (though do read through some of his papers to see if the info you need happens to be in there). His group uses FACS sorting of nuclei to purify cell subpopulations from the brain prior to ChIPseq and have had good success. I expect they've done whole cell FACS as well.
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Old 07-13-2016, 07:37 AM   #3
shldbcrzy1
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Friedrim,

I am planning to run an expt similar to the one that you described (with specific neuronal population) and will fix samples before FACS. Did you get an answer to your question as to the integrity of the DNA interactions ?
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Old 07-14-2016, 12:21 AM   #4
friedrim
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Hi, sorry, but I didnt continue this particular experiment since it turned out to be too difficult to collect enough cells for ChipSeq. Good luck for you!
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