ChIP library prep for TF - ideas? - How best to scale up ChIP for ChIP-Seq (Sep/06/2010 )
Hi Guys, hope you are all well.
I have an interesting problem that I am sure you can all help me with. After getting great (and consistent) results now with my ChIPs, I have decided to take the leap and go with ChIP sequencing using the Illumina platform. I can easily make a very nice library from the input DNA and can get around 150 ng of library DNA from approximately 1 million cells. However as you all know, getting similar results for my TF takes a little bit of scaling up. The problem though is that I use the Magnify Chip kit from Invitrogen and it is a little difficult to scale it up without ending up with mls of DNA, which would need concentrating, resulting in the inevitable loss of precious DNA. The invitrogen kit is usually performed in 100 ul volume (dynabeads) and elutes in 150 ul (magnetic beads) and I usually use 1 million cells/chip. If I did 10 - 20 individual ChIPs I would end up with 1 - 2 ml of DNA. I guess I could then concentrate it but I'm not sure about this. I could also scale up the actual ChIP but this I have not done before and it could be an expensive mistake. I could also elute in less volume but again, I have not tried this and because I use magnetic beads, this would probably not work well.
I guess I am stuck with the first option but does anybody have any other ideas? What do you reckon the best way to concentrate the final product is? Load it all on 1 Qiagen column?
Cheers
Dukey on Mon Sep 6 18:02:47 2010 said:
Hi Guys, hope you are all well.
I have an interesting problem that I am sure you can all help me with. After getting great (and consistent) results now with my ChIPs, I have decided to take the leap and go with ChIP sequencing using the Illumina platform. I can easily make a very nice library from the input DNA and can get around 150 ng of library DNA from approximately 1 million cells. However as you all know, getting similar results for my TF takes a little bit of scaling up. The problem though is that I use the Magnify Chip kit from Invitrogen and it is a little difficult to scale it up without ending up with mls of DNA, which would need concentrating, resulting in the inevitable loss of precious DNA. The invitrogen kit is usually performed in 100 ul volume (dynabeads) and elutes in 150 ul (magnetic beads) and I usually use 1 million cells/chip. If I did 10 - 20 individual ChIPs I would end up with 1 - 2 ml of DNA. I guess I could then concentrate it but I'm not sure about this. I could also scale up the actual ChIP but this I have not done before and it could be an expensive mistake. I could also elute in less volume but again, I have not tried this and because I use magnetic beads, this would probably not work well.
I guess I am stuck with the first option but does anybody have any other ideas? What do you reckon the best way to concentrate the final product is? Load it all on 1 Qiagen column?
Cheers
I think your very last idea is a good one. A colleague in my old lab concentrated his samples down by doing cleanup with a Qiagen MinElute kit. Do you think you would be eluting more than 5µg of DNA?
KPDE on Mon Sep 6 23:50:21 2010 said:
Dukey on Mon Sep 6 18:02:47 2010 said:
Hi Guys, hope you are all well.
I have an interesting problem that I am sure you can all help me with. After getting great (and consistent) results now with my ChIPs, I have decided to take the leap and go with ChIP sequencing using the Illumina platform. I can easily make a very nice library from the input DNA and can get around 150 ng of library DNA from approximately 1 million cells. However as you all know, getting similar results for my TF takes a little bit of scaling up. The problem though is that I use the Magnify Chip kit from Invitrogen and it is a little difficult to scale it up without ending up with mls of DNA, which would need concentrating, resulting in the inevitable loss of precious DNA. The invitrogen kit is usually performed in 100 ul volume (dynabeads) and elutes in 150 ul (magnetic beads) and I usually use 1 million cells/chip. If I did 10 - 20 individual ChIPs I would end up with 1 - 2 ml of DNA. I guess I could then concentrate it but I'm not sure about this. I could also scale up the actual ChIP but this I have not done before and it could be an expensive mistake. I could also elute in less volume but again, I have not tried this and because I use magnetic beads, this would probably not work well.
I guess I am stuck with the first option but does anybody have any other ideas? What do you reckon the best way to concentrate the final product is? Load it all on 1 Qiagen column?
Cheers
I think your very last idea is a good one. A colleague in my old lab concentrated his samples down by doing cleanup with a Qiagen MinElute kit. Do you think you would be eluting more than 5µg of DNA?
Not likely! I think in the order of ng is more realistic. I will try concentrating everything at the end. Will let you know how it goes.
What is the minimum amount of material you can use going into illumina library? Solid ChIP-Seq protcol uses Magnify chip with library protocol for samples less than 10ng.
epi123 on Wed Sep 8 14:53:15 2010 said:
What is the minimum amount of material you can use going into illumina library? Solid ChIP-Seq protcol uses Magnify chip with library protocol for samples less than 10ng.
10 ng is the suggested for the Illumina library too but that is quite a lot of DNA to pull down when you are IPing a TF. So far I have not been able to get enough when doing a single ChIP, but the standard reaction in the Magnify kit only uses 100,000 cells.