A forum for troubleshooting ChIP and related techniques http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 17:27:17 -0800 360 http://www.protocol-online.org/forums/index.php?showtopic=11704
Hope you don't mind my posting a plug for my software here ... hopefully some of you will find it useful. I've written a desktop java software application called MochiView that is a hybrid genome browser and motif analysis/visualization platform. The software is particularly suitable for ChIP-Chip and ChIP-Seq analysis, and has also been used with RNA-Seq and as a stand-alone motif management system.

The MochiView website (link) contains everything you need to get started, including a tutorial and a few sample videos of the software in use (link).

Here is a summary of some of MochiView's features:

[1] Highly customizable plots… display anything from genome alignments to motif matches to bar- or line-graph data on one or more tracks. Open plots are persistent across sessions and you can save your configurations for later use

[2] The plots have smooth scrolling/zooming, lots of interactive elements, and a powerful data browser feature.

[3] Very easy to set up and use. The database is built in to the software, so no fussing around with external databases is required. You can also export your current database and plot configurations to share with other MochiView users.

[4] Motif management and analysis utilities:

• MANAGEMENT: Maintain a motif database (several libraries are available on the website to get you started)
• DISCOVERY: Find new motifs with the built in motif finder (or import MEME and Bioprospector motif searches)
• COMPARISON: Check whether your newly found motifs resemble any other known motifs
• DISTRIBUTION: Search for non-random positioning of motif matches relative to other genomic features such as start codons or instances of a different motif.
• MOTIF ANALYSIS and ROC PLOT: Determine whether your ChIP regions are enriched for a motif relative to a set of control regions
• MOTIF SCORING: Numerous options for motif scoring (LOD score, p-values, w-score) and many different export formats. For example, a few clicks gives you a "gene vs motif" spreadsheet matrix of the motif score for each gene's promoter region.
• MOTIF LOGOS: Export logos of one or more of the motifs in your library
• MOTIF GO TERM ENRICHMENT: Determine whether your motif is enriched in the promoters of genes with common functionality

[5] Location and data analysis and refinement utilities:

• PEAK EXTRACTION: Not a full-fledged peak finder (i.e. no p-values), but this utility does an excellent job of smoothing your ChIP-Chip data and marking regions of a user-defined width under peaks (a useful first step to refining a motif search).
• DATA SMOOTHING: Smooth your data and save as a "Tiled Set" (similar to a UCSC browser wig track).
• DATA REFINEMENT: Numerous filters can refine your data sets (e.g. filter by value or by overlap with other sets of locations).
• LOCATION SET SUMMARY/COMPARISON: Get a detailed description of the genome-wide coverage of one or two set of locations (e.g. ChIP binding regions)
• GENE PROXIMITY ASSIGNMENT: Determine which gene(s) lie near a set of locations (e.g. ChIP binding regions). Can be used in conjunction with a GO term enrichment analysis.
• SET OPERATIONS: Take the intersection, union, or difference of two sets of locations (e.g. find the overlap of two sets of binding regions and remove the portions that overlap ORFs)

[6] Extensive sequence search options, including direct and inverse repeats

Thanks for your time!
]]> Fri, 20 Nov 2009 14:50:44 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11704 http://www.protocol-online.org/forums/index.php?showtopic=11664 I am beginning to work-up the fast ChIP protocol but am having difficulty at one of the first steps - perhaps this doesn't bode well?
I took an aliquot of sheared chromatin before the IP step and extracted the DNA using the chelex protocol given in the Nature Protocols paper for Fast ChIP. When I ran this on a gel, I saw a bright low MW band, and a very faint smear of very high MW. After RNAse treatment the low MW band disappeared, as expected, but there is very little DNA on the gel, if any (if I squint there may be something there, but it could be my imagination).
I used a relatively confluent plate, so should have approximately the 10 million cells/1ml recommended in the protocol. I had reasonably sized cell and nuclear pellets, and small but visible DNA pellets after extraction. Could I have not re suspended my DNA sufficiently in the Chelex?

Can anyone give me some ideas as to where my loss of DNA may have occurred?
Many thanks...
]]> Thu, 19 Nov 2009 00:49:28 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11664 http://www.protocol-online.org/forums/index.php?showtopic=11608
Thanks,

MM]]> Tue, 17 Nov 2009 08:25:53 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11608 http://www.protocol-online.org/forums/index.php?showtopic=11479
]]> Wed, 11 Nov 2009 18:47:01 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11479 http://www.protocol-online.org/forums/index.php?showtopic=11425 I was reading an abstract (ASN) where they studied histone acetylation and corelated that with polymerase density. I thought i need to study the whole promoter region to corelate the level of histone acetylation and a gene's expression. but this one seems to be a good idea in compare to study the whole promoter region.
Please let me know what you guys think and if you have any issues with this type of corelation study.

Thanks,]]> Mon, 09 Nov 2009 07:49:49 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11425 http://www.protocol-online.org/forums/index.php?showtopic=11385 Thu, 05 Nov 2009 13:13:22 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11385 http://www.protocol-online.org/forums/index.php?showtopic=11347
These two antibodies are by far the most popular I've seen in ChIP experiments. I will be using ChIP DNA for next gen sequencing so it's absolutely critical the antibody works well. Does anyone have a suggestion for an alternative, that has been used in a high profile publication, or you have personal experience with?

Thanks.]]> Wed, 04 Nov 2009 07:54:01 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11347 http://www.protocol-online.org/forums/index.php?showtopic=11324 I'm new in ChIP.
I'm using as positive control the acetyl-histone H3 (upstate 06-599), but it gives me a band only for the stimulated condition.
I don't get any band in the not treated condition.
My inputs are fine.
So, I wonder: should Histone H3 always precipitate and be present on the promoter of a gene even if the gene is not transcriptionally active?

I hope some of you can help me.
Thanks!]]> Tue, 03 Nov 2009 09:20:20 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11324 http://www.protocol-online.org/forums/index.php?showtopic=11232
I intend to subsequently use the ChIP DNA for ChIP-seq, but most groups appear to have done the sequencing first and then validate by PCR afterwards, not the other way round. Are there any resources I can use to find regions with a high chance of having a particular histone modification?

I guess one control would be to western blot the ChIP protein-DNA complexes with my original antibody, but that doesn't tell me a great deal I don't already know.

By the way, I'll be using rat cardiomyocytes, if that helps. I hope someone has an answer! Thanks.]]> Thu, 29 Oct 2009 09:42:26 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11232 http://www.protocol-online.org/forums/index.php?showtopic=10976
I'm new to this forum and at the moment I'm trying to get ChIP working with antibodies against MBD2 and MBD3 (of the NuRD complex). Has anyone of you tried this and can recommend an antibody? I already searched literature but even with antibodies used in ChIPs I don't get any signal. Is it for example better to leave out the LiCL buffer? Or is there any "trick" regarding this two proteins?

Thanks for your help]]> Tue, 20 Oct 2009 05:02:31 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10976 http://www.protocol-online.org/forums/index.php?showtopic=10959
I'm struggling to make sense of my ChIP-Chip visual data. Let me precede all of this by saying I've done numerous controls and tests at the sonication, IP, and array steps, all of which appear to give me accurate data. However, when I visualize the final ChIP-Chip data, it appears that there are too many jagged peaks throughout the genome, including some large, wide positive and negative peaks. Still, there are also some textbook ChIP-Chip peaks there as well.

But here's the kicker - I've done three completely separate biological replicates from culture to array and get the same results, i.e., similar peaks between each array (even those positive and negative peaks that don't look quite right). So if the data is repeatable, is it really noise? Or is it indicative of actual binding? I have read that noise can contaminate an array data set and give false peaks, but also that there is the 'appearance' of noise when evaluating global repressors, and that it's actually correct data.

Specs: I'm evaluating a repressor protein in E coli using Nimblegen K-12 WG microarrays.

I don't have the experience with ChIP-Chip data to make final conclusions about this data, so any help from any of you who may have seen this sort of thing before is greatly appreciated. I've attached a small (~90kbp segment of the genome) pic of the three replicates so you can see the repeatability. Note that this is raw data, before the peak-finding algorithm is applied.

Thanks!

Chris]]> Mon, 19 Oct 2009 09:56:01 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10959 http://www.protocol-online.org/forums/index.php?showtopic=10952
I hope you can provide a newbie as me with golden tips solving me problems with ChIP.

I am trying to ChIP on cofactors with a 10 minutes formaldehyde-fixation step. Is 10 minutes too short if you are looking at factors not bound directly to the DNA. I add the formaldehyde (1% final) directly to the media.

I am incubating antibody, chromatin and beads overnight. Is that too long?

Is it sufficient to add Complete as a protease inhibitor to the chromatin or should I also add other inhibitors?

Thank you for your valuable assistance.

Cheers,
Hallenborg]]> Mon, 19 Oct 2009 04:43:42 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10952 http://www.protocol-online.org/forums/index.php?showtopic=10801 I've just realised that I added 10X more salmon sperm to my ChIP than I was supposed too... ... i just can't brain today.

Any idea on what will happen?
Any ideas on how i can fix this?

V]]> Sun, 11 Oct 2009 19:27:03 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10801 http://www.protocol-online.org/forums/index.php?showtopic=10740
I am performing ChIP recently, an odd band always showed up when I ran the agarose gel to check the size of sheared DNA.

Briefly, formaldehyde is added into cells at a final conc. of 1%, for crosslinking for 10 min, and then the reaction is stopped by adding glycine (final conc. 0.125M) for 5min. Cells are washed and lysed by SDS buffer (+protease inhibitor) to get the nuclei. After pelleting, the nuclei are resuspended in IP buffer (+protease inhibitor) and then sonicated. Sonicator S-4000 is used, and protocol is set to pulse on for 1s and then off for 5s with amplitude of 30, for 50 cycles.

2 kinds of cells had been tried, including thyroid cancer cells ARO, and Head&Neck cancer cells FADU.

The 1st (ARO) and 2nd (FADU) figures show the results from same treatment of 2 cell lines.

I thought the unwanted debris present in IP buffer might cause it, so I centrifuged the sample after the 5th sonication, then ran the gel (3rd fig.), however, the band became sharper and brighter.

Could anyone tell me what happened, and how to overcome??






]]> Thu, 08 Oct 2009 03:14:17 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10740 http://www.protocol-online.org/forums/index.php?showtopic=10542 I'm trying to do ChIP and use the resulting DNA to do a qPCR. But the problem is that the amount of DNA in ChIP is very low that I do not get any signal once I do the qPCR. So anybody has suggestions to improve the amount of DNA? I tried doing a PCR of the ChIP product with random hexamers to amplify the signal, but its also in vain. Any suggestions welcome.
Thanks so much!]]> Mon, 28 Sep 2009 13:44:20 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10542 http://www.protocol-online.org/forums/index.php?showtopic=10535
I have started doing histone acetylation study. First we are looking into 1 gene in our 10 samples.
As i dont have that much experience here, i want to make sure i am doing it properly, so need expert's feedback.

I am using millipore kit, 50 microlt of each IP has 1 million of cell equivalent, saving 1% for input. In RT, my total reaction volume is 25 microlt and i am using 12.5 microlt of SYBR along with total 100 nm primer (FP+ RP). I tried different DNA dilutions, like used 2 microlt and then 50 fold, 100 fold, 200 fold dilutions. For all of the these dissociation curves look good. 2.5 microlt of H3K14 acetylated antibody(Millipore) / IP i am using (as this is not purified, no concentration is written).
the Cts i got for one of my samples are : Input 26, IP 25.5, IgG 34.

Do you think, i am doing it properly and the results i am getting are ok? Do you think i need to optimize any other things like antibody amount or something else?
Another question, as i have 10 samples, do i need to do igG for each of the samples?

Thanks,
]]> Mon, 28 Sep 2009 07:59:46 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10535 http://www.protocol-online.org/forums/index.php?showtopic=9910
I am just going to start CHIP expt for Histone acetylation study and this is the first time i will be doing this. There are 4 genes i am interested in. But i have no clue what i can expect about their acetylation level in patient samples.
I was thinking if i have any gene which always shows certain degree of H3K14/9 acetylation, then i can use that as positive control as in that case i already know i will get increased H3K14 acetylation in this gene. I tried to search for article, but could not yet.

I will really appreciate if you can tell me about a positive control i can use in CHIP H3K14 assay.
Thanks,]]> Tue, 25 Aug 2009 08:51:04 -0700 http://www.protocol-online.org/forums/index.php?showtopic=9910 http://www.protocol-online.org/forums/index.php?showtopic=9858 I am having trouble with my ChIP assay...I do end up with a white DNA pellet at the end but after running PCR with primers that our lab uses (others have already optimized the number of PCR cycles), my agarose gel shows no bands, even for my total chromatin samples. Any ideas why this might be? Thanks! Sat, 22 Aug 2009 15:26:44 -0700 http://www.protocol-online.org/forums/index.php?showtopic=9858 http://www.protocol-online.org/forums/index.php?showtopic=9815 joel d nelson,etc
who can send me one copy of this paper, i need this to improve my protocol
my email is bekeforever@hotmail.com
thankyou!]]> Wed, 19 Aug 2009 20:05:38 -0700 http://www.protocol-online.org/forums/index.php?showtopic=9815 http://www.protocol-online.org/forums/index.php?showtopic=9809 Wed, 19 Aug 2009 14:51:17 -0700 http://www.protocol-online.org/forums/index.php?showtopic=9809