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Old 03-08-2011, 06:05 AM   #1
Alex Clop
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Default ChIPseq for histone modifications

Hello there,

I want to do ChIP-seq for H3K4me modified histone and have a native chromatin extraction protocol with micrococcal nuclease step in place. I plan to use the Illumina HiSeq200 for Paired end sequencing of 58bp-long reads.

I understand that the best fragment size range for this approach is 200 Ė 300 bp.

What I donít understand is how a micrococcal nuclease digestion step can be good for ChIP-seq since the resulting fragment size range is quite broad (mono- di and tri- nucleosomal band being abundant) and the sample library prep prior to sequencing requires a very narrow size range.

Moreover, does anyone know whether sonication instead of micrococcal digestion is ok when the subject of study is the histone or its modifications?

Finally, as I want to study T cells from blood, I can obtain around 20 million T cells and am not sure whether this will be enough for sequencing after ChIP.

Can anyone help? Any protocol in place?

Thanks in advance

Alex
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Old 03-08-2011, 09:15 AM   #2
Jon_Keats
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Hi,

My lab is starting to work up a similar protocol for H3K4 and H3K27. Right now we are testing the Magnify-CHIP kit from invitrogen, which claims to use very small cell numbers and recommends sonication. In a couple of weeks I'll be able to update on the success.
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Old 03-08-2011, 09:20 PM   #3
frozenlyse
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We've done lots of chip-seq using sonication (bioruptor) and it works fine - H3K4me3 is especially clean... One of the first steps of the Illumina library prep protocol is size selection where you could just cut out only the mononucleosomes?

Also 20 million cells (in our hands) is plenty to do chip-seq on chromatin modifications - you could get away with 1 or 2 million if the antibody is good enough!
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Old 03-08-2011, 11:33 PM   #4
ETHANol
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Mononucleosomal sized DNA works fine for ChIP-Seq. You have to remember that you add about 70bp to the DNA size with the adaptor ligation. Mononucleosome DNA is 147bp + aprox. 70 bp (Illumina adaptors - check this I'm not sure exactly) gives you about 220 bp of DNA which is exactly what is optimal for Illumina sequencing.

I cross-link with formaldehyde and digest down to mononucleosomal sized DNA and use that as starting material for the IP. It seems you would see nucleosomal positioning better with micrococcal fragmentation then sonication, but sonication works as long as your epitope is still present after x-linking.
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Old 03-09-2011, 06:58 AM   #5
Alex Clop
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Thanks for all your replies, really.

I considered the option of selecting the mononucleosomes but I was resilient as a lot of precious chromatin would then be discarded and the starting amount prior to the library prep is a limiting factor.

As far as I could see following the existing literature, the starting amount of chromatin in ChIP-seq experiments is very variable but many studies used up to 20 or 30 million cells. I was planning to use this large amount to be on the safe side. However, this should not be an issue if two million cells per IP are sufficient. Would it be possible by any chance to have your protocol?

Moreover, to select the mononucleosomal fragments, I can only use micrococcal disgestion, as sonication would give me a large smear not related to nucleosomal bands.

Thanks again

Alex
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Old 02-09-2012, 09:13 AM   #6
mira636
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I am also in need of a protocol that works for 1-2 millions of cells as opposed to 10+ million cells as I am working with FACs sorted cells. I have troubleshooted for 4 months but still cannot recover >10ng for chip-seq.

Would you be able to send me the protocol or a paper reference?

Much appreciated!
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Old 02-09-2012, 09:18 AM   #7
Jon_Keats
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We are using the Magnify CHIP kit from invitrogen and the homebrew protocol from ETHANol above with good success. It uses 1 million cells as input.
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Old 02-09-2012, 02:09 PM   #8
FWOS
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Default Help with Quantification of ChIP Samples

Hi,
Thanks for the info and the protocol. I have some ChIP samples that I wanted to run through the Illumina sample prep protocol, but it seems like they can't be accurately quantified via the nano-drop or the bio-analyzer(HS or 1000). The ChIP protocol used was EpiTect ChIP from Qiagen. After the ChIP prep there seemed to be salt contamination which was seen in the low 260/230 readings from the nanodrop. After running the samples through a minelute column the samples spec @ ~50ng/ul with 260/280 ~1.8 and 260/230 ~2. This seems great, but the bioanalyzer DNA 1000 chip shows no peak for any of the samples. These were IPd with positive and negative control antibodies for histone modification - so the ChIP samples should have ample material, but I had hoped someone might be able to lend their advice.

Has anyone found a good way of determining the concentration of their input into the Illumina ChIP seq sample prep?

Thanks in advance.
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Old 02-09-2012, 02:16 PM   #9
Jon_Keats
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Qubit is what we use but that is because we expect very low concentrations (ie. <2ng/ul). I'd be worried the nanodrop is lying. Is the peak at 260nm or shifted? Alternatively the DNA you have is not fragmented but high molecular weight so not seen on the BioA. The column only retains fragments >100bp typically so if the nanodrop is right you should see it on BioA unless one of the above is the case.
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Old 02-09-2012, 10:32 PM   #10
ETHANol
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Run your input on a 2.2% agarose gel to determine the fragmentation (do NOT run too much - most people overload and you do not get an accurate representation of fragment size). Then use the QubeIT HS DNA assay kit to determine the concentration.

I don't think the Bioanalyzer or the Nanodrop are good methods for DNA quantitation. I have no hard evidence on this but I still would recommend against using them.

I'm using AMPure XP beads to purify my ChIPs now and works really well. Excellent recovery of fragments over 100 bp and small quantities of DNA (5 ng). You can see what I do at the end of this protocol.
http://ethanomics.wordpress.com/chip...aris-protocol/
I haven't tried the AMPure beads with native ChIP yet but I think it should work even with 140 bp mono-nucleosomal fragments.
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