ChIP Method Discussion

Formaldehyde cross linking problem!!!

Thu, 09 Feb 2012 12:38:15 +0000

Hi,

I am trying to optimise ChIP protocol and not able to go pass first step of cross linking!

I am collecting 3-4g plant tissue and crush it in liquid nitrogen. Followed by either storing them in -80 or use it to corsslink in a buffer containing 1% formaldehyde.

I have tried 1% formaldehyde to corsslink my samples (2.5, 5 and 10 minutes). Interestingly, I do see DNA when I De-crosslink but in (IP) control samples which are not de-cross link I dont see even a faint DNA band. Controls without formaldehyde gives good band in de-crosslink or non de-crosslink. This has led me to understand that the samples are over corsslinkned.

1) I am wondering if any one has done cross linking with samples which are first powdered in liquid nitrogen?

2) is 1% formaldehyde ( 37% formaldehyde containing methanol is used) concentration too much for powdered sample?

Cheers for any help in advance!

I'd like your advice on NIH 3T3 sonication

Tue, 07 Feb 2012 23:37:28 +0000

Good day, (please excuse my english, it's not my first language)
I recently started my master less than a year ago and I'm currently trying to do ChIPs on NIH 3T3 cells. My lab's old protocoI requires 25 minutes of sonication and doesn't give really nice chromatin fragments. I decided to try different conditions and I was able to get very nice chromatin fragments (I think so) by sonicating 50e6 cells in 1 ml of 1% SDS nuclei lysis buffer in the diagenode bioruptor (2*5 cycles of 30s on/30s off). Here's the timecourse I did.
However, I would need to dilute 1:10 my chromatin to IP in 0.1% SDS. That would mean 3.2 ml for 16e6 cells, wich is way too much. I'd like advices from people who usually do ChIP on NIH 3T3. What is your sonication buffer? How long do you have to sonicate? How many cell do you sonicate at a time and in what volume? Feedback would be greatly appreciated.
Thank you very much!
Jo�l

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Untreated sample as negative control for ChIP-on-chip?

Thu, 12 Jan 2012 13:41:52 +0000

Hi,

I would like to do some ChIP-on-chip experiments using Affy promoter arrays to check for binding of a TF after treatment.

People at Affy say that I will need to run at least 2 chips per condition (one neg ctrl IP sample (IgG or No Ab) and the Ab against the POI), to correct for unspecific IP.

Now I was thinking, since my TF is only activated after treatment, I already don't expect to see much specific enrichment in untreated samples. So do you think I can just use the untreated samples as neg controls and express my data as enrichment relative to untreated samples? Or is it always necessary to have a neg. control from every single ChIP sample?

Cheers,
Roel

ChIP data normalization

Sun, 01 Jan 2012 06:11:42 +0000

Hi, I am wondering how best to normalize my ChIP-qPCR data.

I did H3K9me3-ChIP on samples from two different tissues.to compare the %Input at my gene of interest. One of the tissues showed reproducibly higher levels of H3K9me3 than the other tissue (n=4 independent replicates), but the interesting thing is that the positive control primer in the heterochromatin also gave consistently higher H3K9me3.

Now, the question is whether the ChIP values are indeed higher in both the gene of interest and the positive control primer (true positive), or whether the consistently higher values are caused by a systematic error in sample handling (false positive). I am thinking whether I should normalize my %Input with respect to the positive control primer to make the positive control score constant, and then read the new %Input at my gene of interest.

Do your positive control primers always have very similar values across biological replicates?

Or do people recommend any other methods of normalization?

help on % input calculation please!!

Wed, 14 Dec 2011 17:29:36 +0000

Hi,

Here's my problem: I just performed a ChIP experiment in which

1. After cross-linking and sonication I took 5 ug of DNA for input and IPs samples.
2. After, IP, cross-linking reversal and DNA purification I eluted DNA on 30 ul (all samples including input).
3. I performed a qPCR raction using 1 ul of input and 3 ul of IPs samples.

Questions are:
1. How do I calculate the % input?
2. Is it absolutelty necessary to tun a standard curve whith input dilutions or can I just aplly the 3,3 Ct differences over 10 fold dilutions??!

Thanks in advance I'm stuck on this one for so long now!!

Real-Time PCR using ABI Fast Sybr Green chemistry

Tue, 13 Dec 2011 15:35:08 +0000

Hey people,
I just started setting up a ChIP protocol and this forum has been of great help. I have a "small" problem with my real-time PCR. Although end-point pcr is working fine, real-time PCR is not. I am using the ABI Fast Sybr Green and do a two-step PCR [95oC for 3secs and 60oC for 35secs] with an initial denaturing/taq activation step of 20secs at 95oC. The problem is that my Cts are over 45, which is not comparable with the end-point PCR results where 22 to 28 cycles are enough for different primer pairs to get the linear PCR phase.
I suspect that Fast Sybr Green needs a bit of a boost. Is anyone else using this kit to spare a tip? If not, could anyone suggest a sybr green kit that has been working for them?
Thanks!

GP

ChIP on episomal DNA

Thu, 08 Dec 2011 11:59:26 +0000

Hello all,

I am working on non-viral vector DNA and trying to figure out why the transgene is silenced in our vectors. To that end, I'm trying to come up with a cell culture model.

I was wondering if anyone's had a stab at doing ChIP on non-integrated, episomal forms of plasmid DNA?

Do you start with the Hirt method to isolate your extrachromosomal DNA first and then proceed to ChIP as 'standard' (haven't picked a particular protocol yet)?

Would this pDNA be much more susceptible to sonication? (I've read the fab article on here by Dukey about sonication vs MNase digestion- too bad MNase digestion still requires sonication though).

Many thanks in advance for your help!

ChIP assay using heart tissue?

Tue, 29 Nov 2011 23:38:48 +0000

Hello, I have never done ChIP assay before, thus my question may be very ignorant..I am planning to do ChIP assay using heart tissue, and immediately found two problems:
1. how to lyse/homogenize heart tissue? it is pretty tough tissue, not well homogenized by glass homogenizer..

2. I bought enzyme-digest based ChIP assay kit(active motif), by thinking enzyme digestion may be easier than sonication, but found it in the protocol that I can't use column to purify DNA but to use chloroform/phenol method and suggested protocol is.....very tricky and time consuming. Is column purification really NO NO in enzyme digested based ChIP?

3. any simplified ChIP method to suggest? Even with kit, it takes forever to do so...I have quite a bit of samples thus the simpler/quicker the better.

4. I am planning to perform qPCR at the end to quantitate. Then what is the criteria to design the primer for ChIP-qPCR? Usually putative response element is <20bp, then PCR should cover only that region? Or make it 100-200bp around it? Any online tool to do so?

Thank you so much for your advice in advance.

:-)

Sung

ChIP amplification problems

Wed, 23 Nov 2011 13:01:47 +0000

Hi there,

I have a problem with amplifying my ChIPs using qPCR. I originally used diagnostic PCR and was only able to amplify up my Histone H3 positive control. Since swithcing to qPCR I was able to get results with one of my experiments and can view differences in binding by gel electrophoresis.

However, with the next set of experiments, I've had a strong band pop up in my IgG lane with 2 different primer sets that is the same intensity as all the other bands there. I've tried fresh SYBR green, primer dilutions, everything and still had this problem. For another primer set, I've then tried stopping the reaction before the curves level off so I can measure the differences, but alas I had lots of smearing in all my lanes. I've also tried diluting down the DNA 10 and 100-fold and I still got a band in my IgG and nothing in my Histone with the same primer set.

I am ChIPPing with IgG, Histone H3, Sprouty2 and HIF1 alpha on various regions of the VEGFA promoter.

I followed the Millipore protocol and used a nucleospin kit to extract the DNA.

Anyone have suggestions as to my qPCR woes?

I would really appreciate the help,

Thanks,

Dave

Highly Variable DNA yield from Neutrophils lysed & sheared for ChIP

Thu, 03 Nov 2011 18:24:13 +0000

Hello All,

I am using the MAGnify ChIP kit on human neutrophils, freshly isolated from peripheral blood. Because I am doing ChIP-seq, I need DNA fragments between 100-300 bp in size. The cells are cross-linked for 10 minutes in 1% formaldehyde. I am lysing and subsequently shearing 2 X 10^6 neutrophils in 100 uL of lysis buffer using a Covaris S2 for 12 minutes with the following settings:
Duty cycle : 5%
Intensity: 2
Cycles per burst: 200
Cycle time : 60 seconds
Cycles:12
Tempterature: 4C
Power Mode: Frequencing Sweeping
Degassing mode: Continuous

My problem is that while I follow the MAGnify Protocol closely, I have large variability in the yield of DNA following shearing, based on analysis on a 2% agarose gel of via analysis on an Agilent dsDNA 7500 chip. For example, a "good" shearing of 2 X 10^6 neutrophils when diluted 1:10 in TE to analyze on the 7500 chip will give a concentration in the range of 10-12 ng/uL for the 100-300 bp region. A "bad" shear run, again when diluted 1:10 in TE and run on the 7500 will give between 1-2 ng/uL in the 100-300 bp region.

Does anyone have any thoughts about why this is happening? I keep the cross-linking and shearing conditions the same between experiments. Sometimes I snap-freeze the cross-linked and lysed cells before shearing them the next day and this hasn't conclusively influenced things either way.

Any thoughts about factors that influence DNA yield from shearing and/or working with neutrophils for this experiment would be highly appreciated.

Cheers!

ChIP-PCR: Amplification in IgG negative control

Thu, 27 Oct 2011 14:29:42 +0000

Hi,

I have a basic question about PCR after ChIP.
So while trying to do this ChIP-qPCR, yes qPCR, I got difference between Ct values of my target antibody IP and IgG IP as 2, so 33 and 35.
But when I ran this product on a gel, I got bands in both target IP and IgG IP, is that normal, since I am looking at the end product of PCR cycles after 50 cycles ?

Thanks !

ChIP from mouse tissues with antibody raised in mouse

Wed, 26 Oct 2011 04:46:11 +0000

Is there any reason why you cannot ChIP from mouse tissue with an antibody raised in mouse?

Washing buffer and use of Protease inhibitors

Fri, 14 Oct 2011 15:50:06 +0000

Hi, I have a question regarding the washing step in ChIP

The buffer for washing the immunoprecipitated DNA-bead complex should contain protease inhibitors? I guess that at the first wash there's still a significant amount of proteases from cell lysate but at the following washings I believe that there's no need to use protease inhibitors or use a lower amount. Please tell me how you do it. Thanks.

SDS Precipitation

Wed, 28 Sep 2011 15:38:30 +0000

Ok I have a major problem with my ChIP assay. After I've reversed the crosslinks and done the proteinase K step, I inactivate the proteinase K by boiling for 10 minutes.

Then I add 5 times the volume of binding buffer from a pcr/gel purification kit. However, a misty precipitate forms (which I assume might be SDS) not matter how much buffer I continue to add. The first time I did this was using a QIAGEN kit.

A friend recommended using a nucleospin clean up kit using a buffer which specifically gets rid of the SDS. I tried this and I still have some crud in my eluates and thus my PCR shows diddly squat!

Is anyone aware of this problem occurring or am I doing something ridiculously stupid? Thanks.

JMJD3/UTX antibodies

Mon, 19 Sep 2011 18:37:32 +0000

Anyone have any recommendations? I know there are several out there, but I was curious as to their performance.....particularly in ChIP and IF. Any help is greatly appreciated.

Proteinase K step

Thu, 08 Sep 2011 19:16:47 +0000

I'm using the Millipore protocol. After reversing the crosslinks with 5M NaCl, I then proceed by adding 0.5M EDTA, 1M TrisHCl pH 6.5 and 10mg/ml proteinase K to my samples, then heat for 1 hour for 45 minutes. After that the protocol states that you carry on with DNA extraction via column spins or phenol/chloroform.

However, there is no mention of needing to denature the proteinase K.

Is there any need to do this?

Thanks

Cell lysis before shearing

Mon, 05 Sep 2011 04:13:30 +0000

Hi there,

I am trying to do ChIP on mouse CD4 T cells, but currently suffering inefficient DNA shearing issue.
I use Millipore's magna-ChIP kit and its protocol mentions to perform cell lysis and nuclei isolation before shearing.
Since having an issue with shearing, I checked with microscope if cells were lysed well after the lysis step. And the result was they were all intact even after several swelling and lysis atempts.
My protocol uses 15 minutes of cell swelling with a buffer containing no NaCl, and additiion of NP-40 final concentration 0.5% and hard vortexing.
How could I solve the issue?
Thank you in advance.

ChIP-Seq with HA-tagged protein

Thu, 18 Aug 2011 22:40:30 +0000

Hi everybody,

I am trying to define site-specific and off-site binding of a DNA binding protein in human cells (at this point HEK-293 and K-562 cells).

The protein of interest is not expressed naturally in human cells, i.e. it has to be expressed heterologously. Furthermore, at this point there is no antibody available for the protein. Therefore I am using a N-terminal HA tag for ChIP with a ChIP-grade antibody.

Here's a quick run down of my protocol:
- transfect (HEK-293) or transduce (K-562) cell with expression vector
- growth for 48h
- fix cells 5x10exp7 with formaldehyde (1% for 10min) quench with Gly
- QC: WB with the a-HA antibody that I use as well for ChIPing. Protein expression can be verified under a fluorescent light microscope or by FACS since my protein is linked via a T2A sequence to mCherry.
- cell lysis and sonication on a Covaris AFA instrument
- binding of the HA-tagged protein to the a-HA antibody immobilized on magnetic beads (protein G) ON at 4°C.
- wash, crosslink reversal and elution
- ProteinaseK and RNase treatment
- QC: size distribution of the sheared DNA
- DNA end repair, addition of A bases, ligation of (barcoded) sequencing adapters and PCR amplification
- QC: amplification is done on a light-cycler using SYBR-green to monitor the emergence of amplification products.
- PAGE purification of the amplified libraries.
- QC of the libraries on an Agilent Bioanalyzer; I aim for fragments of 200-400bp.
- 76bp SE Illumina sequencing

I get 20-60 mil reads that I map back to GRCh37/hg19 using bowtie or bwa. Peak calling is done with MACS.

My problem is, that I get a LOT of background. One reason for this is already the low percentage of cells that initially express my protein. Based on mCherry expression I can say that it's only about 1/3 of the total number of cells that light up under the microscope. In order to alleviate this problem I've put together a new expression plasmid that has a selection marker and is Tet inducible so that I can hopefully increase this number. I didn't get to try it out yet though. Cell sorting is not an option because of the large number of cells.
On that note, does anybody have a rough idea of the number of molecules/cell of a protein one needs for a reliable ChIP-Seq experiment?

Besides the amount of protein I suspect that the main problem is the a-HA antibody that I am using for the ChIP. Has anybody already used the HA-tag for ChIP?
Which tags might be better choices in this setting? I am considering to switch to another tag, namely GST or His. I've seen a study in S. cerevisiae that featured a myc-specific antibody for ChIP but I guess if I use that in human cells I will pull down all the c-myc targets.

It is not known if and where my protein of interest binds in the human genome - that's exactly what I am trying to figure out. As long as I can say with confidence that there are no binding sites I'd be happy with that. As positive control I use a known binding sequence for my protein that I inserted into the HEK-293 genome (I don't havea positive control in K-562 cells).
I do not see this positive control after peak calling. At this point I fear that potential peaks might either not be visible because of insufficient enrichment (either caused by the low number of protein molecules per cell or inefficient pull-down of bound sequences) or because they are covered by the strong background.

I'd appreciate any suggestions,

Wonko

ChIP-qPCR gives odd amplification plot

Tue, 02 Aug 2011 17:50:29 +0000

Hi all,
I was hoping for some help with a problem analyzing my ChIP-qPCR. My amplification curves are shaped oddly and sometimes sigmoidal. When I look at raw fluorescence, I see that it starts to come up above background in early cycles, levels off, and comes up again. When I run a gel on the product, I only see my intended amplicon.

I think it may have to do with my fragment size (200-600bp), but am not sure what I can do about it during analysis. I've been trying to fiddle with the baseline settings, but have not been terribly successful. Any advice would be appreciated!

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ChIP help- Ab from different sources

Tue, 02 Aug 2011 16:26:41 +0000

Hi,

I am attempting to do ChIP for my protein of interest. However i have multiple issues to address and would seek some help. I over express my protein and have tagged it with V5 epitope. (protein is functional and has been tested). I am attempting to IP using antibody for V5 which is conjugated to agarose using the Diagenode kit.

Q1. Is it sufficient to use unconjugated isotype + beads (equal conc. of Ab as V5) as one of the negative controls?

Q2. I use GAPDH promoter (Diagenode kit) primer as negative control. I see an increase in ct for V5-Ab ip compared to IgG, Is that normal?

Q3. I do not know the binding site for my protein to DNA. My hunch is that it may be co-partnering with already known promoter element so i designed primers for it and observed a 1.5 to 2 Ct difference comparing V5 IP to IGG. What difference is considered acceptable?

thanks
Sam