ChIP seq sample prep for Illumina

[cjohns]

Here is what I found on our Chip-Seq:

Neither Nano-Drop nor bioanalyzer nor Pico Green quantitation of our IP products agree so we stopped trying to quantitate them. We use 1/3 of the IP product for the sample prep protocol (the same amount we used for Chip-chip and it gives us good results)

The gel purification step is much trickier than their protocol states. If you run the gel for 60 min as they state and cut out 200+/- 25 bp you will get a huge amount of adapter complexes in your final sample that will show up as a big peak at about 120bp on bioanalyzer results after amplification. They will also make up about 50% of your sequences on the machine. What we do is run a much longer gel for at slow speeds for 5+hours and cut out 180-300 bp and then continue with protocol as is.

Amplifying after size selection has not led to any perceivable contamination by ladders (of course, leave empty lanes between samples and ladders).

[Chipper]

Hi, I have not done the sample prep myself and not seen the protocol but I thought size selection was supposed to be after enrichment as it makes more sense to me. Otherwise the risk of contamination by ladders or other saples increases and you will probably lose a lot of fragments during size selection. This protocol uses amplification first: http://www.genomecenter.ucdavis.edu/...brary_prep.pdf

[athos]

thanks Chipper. Can I just ask - do you normally follow the Illumina sample prep protocol as is, or do you ever do the size selection after the enrichment PCR?
Cheers.

[Chipper]

Yesy, we do but it does not tell you much since you do not know what is contaminaition and or background DNA. Usually PCR or qPCR will be more informative, if you have a good positive control.

[athos]

Hi Everyone, I'm hoping someone can give some advice on ChIP enriched DNA. Does anyone try and quantify this by nanodrop/picogreen before starting the Illumina sample prep protocol (the protocol recommends starting with approximately 10ng DNA)? We have had difficulty in quantifying samples reliably. Is anyone doing ChIP seq regularly that can help?
Thanks.