Support | FAQs | LowCell# ChIP kit

Frequently asked questions


General FAQs


Differences with sample types


Amount of starting material


DNA-protein cross-linking


Cell lysis and Chromatin shearing


Gel analysis questions


Antibodies


Kit controls


Purification


Post-immunoprecipitation yields


Questions regarding general applications: microarrays, sequencing, PCR


Amplification of immunoprecipitated DNA


General FAQs

What is a tube clap (as referenced in the LowCell# ChIP kit) and where can I buy them?
The common name for tube claps is “microcentrifuge tube holder.” This holder prevents the tube from opening during the boiling. You can purchase these tube holders from LabScientific or Sigma. Alternatively, you can wrap the tops of the closed tubes with parafilm.
For ChIP with tissue, what machine can I use for tissue disaggregation?
The protocol "ChIP from tissues with the LowCell# ChIP kit is optimized with the Medimachine from Becton Dickinson, but you can use other machines for tissue disaggregation. You may have to adapt the amount of time to process all the tissue with other equipment, so it is best to check your cell suspension by microscope.
Is it possible to freeze the samples at some steps of the protocol?
  1. Pellets of formaldehyde fixed cells can be stored at - 80°C for at least a year.
  2. Sheared chromatin can be stored at - 80°C for months, depending on the protein of interest to be ChIP’d.
  3. Purified DNA from ChIP and input samples can be stored at -20°C for months. Nevertheless avoid multiple freeze/thawing.

Differences with sample types

Which cells were used for the validation of LowCell# ChIP protocol?
HeLa, NCCIT 293T, Chondrocytes, P19, ASC (adipose stem cells) and Kerationocytes have been used to validate the Magnetic ChIP protocol.
Can the kit be used for nuclear preparations that were derived from frozen samples?
If the experimental tissue was not frozen in a cryopretective medium, it is possible that the nuclei will be fragmented. Thus, Diagenode recommends working with fresh tissues. Samples can be frozen after nuclei lysis and chromatin shearing at -80°C for several months and yield good results. Alternatively it is possible to fix the tissue and then to freeze the samples.
I would like to use the LowCell# ChIP kit with the fertilized eggs. The problem is that each time I am collecting very small amounts of eggs, so I need to pool down several samples to have the amount necessary for ChIP experiment. My question is how can I preserve the samples after cross-linking without freezing them?
We have never tested the stability of fixed cells at 4°C, we always freeze them or directly sheare the chromatin. You might try to keep fixed eggs in PBS with added proteases inhibitors at 4°C for some days but there is a risk of chromatin degradation. The best way it would be to test it yourself: compare the results obtained from this variant with the results from fresh material to be sure that it works. We would recommend to freeze the samples or to go directly for chromatin shearing and continue with the protocol of LowCell# ChIP kit.
Can the kit be used for ChIP with plants?
Preparation of plant material for Chromatin Immunoprecipiation differs essentially from preparation of culture cells, so Diagenode has adapted the protocol of the LowCell# ChIP for this application. We recommend therefore to follow the first steps of a ChIP protocol optimized for plant material (www.diagenode.com/media/downloads/documents/protocols/ChIP_from_Plant_Material_with_the_LowCell_ChIP_Kit_protocol.pdf) and then to go ahead with the LowCell# ChIP.
How can I adapt the kit to work with the tissue?
Diagenode has adapted the kit for this application. When working with tissue start your experiment using the protocol for chromatin preparation from tissues (www.diagenode.com/media/catalog/file//Protein_extraction_from_Tissues_and_cultured_cells_protocol_standard_Plus.pdf) and constinue your assay with LowCell# ChIP kit.

Amount of starting material

What is the maximum amount of cells I can use per immunoprecipitation?
Up to 1 million cells can be used per immunoprecipitation, but some modification of protocol is necessary. For the details please, look at the document: “LowCell# ChIP kit with 1milion cells”
How to compare unstimulated and differentiated cells in ChIP, if you use the same amount of cells in the beginning? For instance, not all of human monocytes will differentiate into macrophages, therefore we have different amount of starting material for ChIP! How can I solve this problem?
You can estimate cell amount by quantifying DNA in cell lysate (OD 260) keeping in mind that 1 somatic cell has 6,16 pg of DNA. Moreover, we count cells after cell collect taking an aliquot of suspension.

DNA-protein cross-linking

In the protocol you propose 10 min of fixation in 1% formaldehyde. To better optimise my protocol can I change the time of fixation or the concentration of formaldehyde? Why this step is so important?

Very short cross-linking time can lead to DNA loss; very long time can lead to elevated background, therefore the optimal cross-linking time should be found empirically as maximal specificity and efficiency of ChIP. To strong cross-linking can affect both efficiency of chromatin shearing and efficiency of specific antigen immunoprecipitation. Shorter cross-linking times (5 to 10 minutes) and/or lower formaldehyde concentrations (1%, weight/ volume) may improve shearing efficiency while, for some proteins especially those that do not directly bind DNA, this might reduce the efficiency of cross-linking and thus the yield of precipitated chromatin. It is possible to optimize the fixation step by testing different incubation times: such as 5, 8, 10, and 15 minutes. Do not cross-link for longer than 15 minutes as cross-links of more than 15 minutes can not be efficiently sheared.

Do not forget to stop cross-linking by quenching formaldehyde with 125 mM glycine during 5 minutes and washing with PBS.

In your protocol you suggest to fix the material at RT. Why not at T=37°C, like in some other protocols?
It is possible to fix at both RT or 37°C keeping in mind that fixation is faster at 37°C. The important thing is to keep the same conditions from one experiment to another in order to avoid over-cross-linking.
The conditions of centrifugation proposed in the manual (Step 2, point 15: is 470 xg) will be sufficient to centrifuge monocyte suspension in order to get a monocyte pellet?
We have not tested monocytes. I suppose it should be enough. Otherwise, you can increase the centrifugation rate to get pellet.

Cell lysis and Chromatin shearing

Should Buffer B be clear or cloudy before use?
Buffer B should be clear before use, as this indicates even detergent concentration. If the buffer becomes cloudy, you may leave the Buffer B at room temperature for some time and rub the bottle between your hands, if necessary.
Why my cells were not completely disrupted after the lysis?
Do not use too many cells per amount of lysis buffer (W/V). Follow the instructions in the protocol (e.g.: 1 million cells or less/ 130 µl of complete Buffer B, see STEPS 2 and 3). Avoid over-fixation which can provoke the difficulties during the lyses step (cells will not be lysed efficiently).
Is it necessary to add the protease inhibitor to the lysis buffer?
It is better to do it, because protein degradation can occur during lysis.
In order to establish chromatin-shearing efficiency protocol, I need detailed information about the components of Buffer B. Could you please provide me with it?
Unfortunately, Diagenode does not provide the composition of buffers. Note anyway that the efficient cell lysis and chromatin shearing require SDS (0,7-1 %).
How can I check the shearing efficiency working with 5000 cells per IP?
To check the efficiency of shearing in agarose gel you need an equivalent of 100.000 cells. It is not possible with lower amounts of cells. The best way is to optimize the shearing on higher amount of cells before doing ChIP on low amount of cells. If it is not possible the others known way to check the shearing efficiency are:
1. PCR-based assessment as described in Genome Biol. 2009 Feb 10;10(2):R13. by Dahl JA, Reiner AH, Collas P (supp data).
I am trying to establish the protocol for best shearing efficiency results for human monocytes. As a results I got the fragments in the range of 200-400 bp, but also >1 kb. What would you recommend me for the next step?
  1. You can increase the time of sonication till 20 cycles instead of 16.
  2. You can try to decrease the time of fixation. Our in house experience show than even 15-20 min of fixation leads to no-shearable chromatin!
  3. Be aware also of quality of FA used (formaldehyde suitable for ChIP will be available soon at Diagneode) and always use the fresh one.
  4. Very important: to stop the fixation using the glycine and washing twice with PBS.
Is it possible to store the 20 µl pellet-suspension of sheared chromatin at -80°C and add the Buffer B the following day (Step 3. point 18)?
Once fixed and washed, the cell pellet can be kept frozen at -80°C at least for some weeks. Avoid defrost.
Should the combination of Buffer B with the sonicated cells be cloudy or clear before proceeding to the immunoprecipitation?
The sheared chromatin must be clear before proceeding to the immunoprecipitation. Thus, you can add Buffer A or rub the tube between your hands to make the sample clear.
After sonication, the 130 µl sample (Buffer B + sonicated cells) is cloudy. Should I add the 870 µl Buffer A whilst the sample is cloudy? Or should I first make it clear by rubbing it between my hands?
You can add Buffer A on cloudy sample or make the sample clear by rubbing it between your hands. It is the same. But after dilution, the sheared chromatin must be clear for the immunoprecipitation.
Can I use in LowCell# ChIP kit the sheared chromatin prepared with Shearing ChIP kit?

Yes, it is possible with small modifications: 30 µl of shearing buffer (from Shearing ChIP kit) is added per million cells. After shearing, transfer to a new tube the amount of chromatin needed (corresponding to a determined cell equivalent), and add Buffer B from the LowCell# ChIP kit to reach a total volume of 130 µl as shown below:

  1. Add 30 µl of sheared chromatin to 100 µl of Buffer B (final: 1,000,000 cells /130 µl).
  2. Add 3 µl of sheared chromatin to 127 µl of Buffer B (final: 100,000 cells / 130 µl )
I would like to use Shearing Optimisation Kit to optimize shearing conditions for chromatin which will be used in LowCell# ChIP kit. But according to the protocol of this kit I need to use 6 mln cells! Is it possible to use this kit and to decrease amount of cells?

You can definitively use the Shearing Optimisation Kit and decrease the amount of cells: Shear your samples in 130 µl of Buffer D (instead of 400 µl as it is written in the protocol of Shearing Optimisation Kit). After shearing use the LowCell# ChIP kit staring by dilution step: add 870 µl of complete Buffer A and continue with the protocol.

In this volume = 130 µl you can decrease the amount of cells but the limit is 100,000 cells, because this is a minimum which can be visualised on the gel.

Can I use sheared chromatin prepared with my own protocol?

Yes, it is possible, but: When using your “in house” protocol: check the composition of the shearing buffer that you use: as it should contain between 0.75% and 1% SDS, EDTA (1-10 mM) and/or EGTA (0-0.5 mM) and should be at pH 7.6 - 8.0. The sheared chromatin is to be diluted in the Buffer A and Buffer B prior to immunoselection:

  1. Add Buffer B to sheared chromatin (final: 1,000,000 cells / 130 µl).
  2. Add Buffer B to sheared chromatin (final: 100,000 cells / 130 µl).
What is the protocol for chromatin shearing using the LowCell# ChIP kit and the sonicator Branson 450? The conditions: 6x10 sec at 30% of power would be sufficient?
At Diagenode we recommend the sonicator Bioruptor, which we know very well. We have no experience with Branson 450 sonicator and we can not suggest the conditions of sonication with this machine. Probably 30% of power is too low for chromatin (with Bioruptor we use the high power). The researcher has to optimise the sonication by him self controlling the shearing efficiency on agarose gel. When adjusting the volume, buffer B should be added (lysis buffer used for shearing but not the buffer A, used for dilution of chromatin before IP).
What are the shearing conditions for a probe sonicator?
The efficiency of sonication will depend on the type of sonicator which you use, so it is necessary to optimize the conditions for your equipment. Try different conditions starting by the sonication for 3 x 30 seconds on ice. Allow a 30 seconds pause on ice between each pulsing session. Take care to avoid foaming. If the size of sheared chromatin is to big try to sonicate longer time.

Gel analysis questions

What can be used as negative ChIP control?
  1. Non-immune IgG fraction from the same species the antibodies were produced in.
  2. Incubation with beads, which were not coated with antibodies, could also be used as a negative ChIP 3) Use one antibody in ChIP and the same antibody that is blocked with specific peptide.
How many negative ChIP controls are necessary?
If multiple antibodies - of the same specie - are to be used with the same chromatin preparation then a single negative ChIP control is sufficient for all of the antibodies used.
How do I choose an antibody for ChIP?
Be aware that your antibody is ChIP –grade. If you ChIP-grade antibody against your target does not exist you can try to use IP-grade antibody.
Why is my antibody not working in ChIP?

There are several reasons:

  1. Antibody-antigen recognition can be significantly affected by the cross-linking step resulting in loss of epitope accessibility and/or recognition.
  2. Use only the antibody of high quality, ChIP-grade.
  3. The best ratio: antibody per amount of chromatin has to be used in ChIP assay.
  4. Do not forget to dilute the sheared chromatin (which contains the SDS) before immunoselection incubation step as it is written in the manual.
What is the amount of antibody per ChIP to use?
It should be determined empirically for each target and antibody. For abundant proteins, like histones, use 1 to 2 µg of affinity purified or monoclonal antibody per IP. For other targets, use up to 10 µg per ChIP. To ensure efficient IP it is important to have an optimal ratio between the amount of chromatin and the amount of antibody. More antibody (or less chromatin) can be required in case of low affinity to antigen or high abundance of target protein (e.g. histones). Insufficient amount of antibody can result in low efficiency of ChIP whereas large excess of antibody might lead to lower specificity.
Is the negative control IgG from rabbit in the LowCell# ChIP Kit crude serum or purified IgG?
Both IgG controls (rabbit and mouse) are purified.
Using the LowCell# ChIP kit I got high values for IgG. Could you explain why?
  1. To be correct in interpretation it is important to use the same amount of IgG as the antibody.
  2. Note also that the efficiency of chromatin shearing can influence background: chromatin that is not enough fragmented give high background (fragments of 1000 bp and more). You should work with average size of fragments: 300-600 bp.
  3. Be also aware that there is no cross-contamination between tubes (IgG and IP). Pay attention when open the tubes, do it softly not to transfer a liquid from IP tubes to IgG control.
  4. Additionally it is possible to perform one more washing with the buffer A and C.

Antibodies

What can be used as negative ChIP control?
  1. Non-immune IgG fraction from the same species the antibodies were produced in.
  2. Incubation with beads, which were not coated with antibodies, could also be used as a negative ChIP 3) Use one antibody in ChIP and the same antibody that is blocked with specific peptide.
How many negative ChIP controls are necessary?
If multiple antibodies - of the same specie - are to be used with the same chromatin preparation then a single negative ChIP control is sufficient for all of the antibodies used.
How do I choose an antibody for ChIP?
Be aware that your antibody is ChIP –grade. If you ChIP-grade antibody against your target does not exist you can try to use IP-grade antibody.
Why is my antibody not working in ChIP?

There are several reasons:

  1. Antibody-antigen recognition can be significantly affected by the cross-linking step resulting in loss of epitope accessibility and/or recognition.
  2. Use only the antibody of high quality, ChIP-grade.
  3. The best ratio: antibody per amount of chromatin has to be used in ChIP assay.
  4. Do not forget to dilute the sheared chromatin (which contains the SDS) before immunoselection incubation step as it is written in the manual.
What is the amount of antibody per ChIP to use?
It should be determined empirically for each target and antibody. For abundant proteins, like histones, use 1 to 2 µg of affinity purified or monoclonal antibody per IP. For other targets, use up to 10 µg per ChIP. To ensure efficient IP it is important to have an optimal ratio between the amount of chromatin and the amount of antibody. More antibody (or less chromatin) can be required in case of low affinity to antigen or high abundance of target protein (e.g. histones). Insufficient amount of antibody can result in low efficiency of ChIP whereas large excess of antibody might lead to lower specificity.
Is the negative control IgG from rabbit in the LowCell# ChIP Kit crude serum or purified IgG?
Both IgG controls (rabbit and mouse) are purified.
Using the LowCell# ChIP kit I got high values for IgG. Could you explain why?
  1. To be correct in interpretation it is important to use the same amount of IgG as the antibody.
  2. Note also that the efficiency of chromatin shearing can influence background: chromatin that is not enough fragmented give high background (fragments of 1000 bp and more). You should work with average size of fragments: 300-600 bp.
  3. Be also aware that there is no cross-contamination between tubes (IgG and IP). Pay attention when open the tubes, do it softly not to transfer a liquid from IP tubes to IgG control.
  4. Additionally it is possible to perform one more washing with the buffer A and C.

Kit controls

What should I do if my negative controls used in the immunoprecipitation (IgG and only beads not treated with any antibody) are positive for c-fos in my qPCR?

It looks like there is a problem with cross-contamination.

  1. Make sure the stock solution of beads is not contaminated.
  2. Run a negative control for the PCR (sample without template DNA) to assure the contamination did not occur with the PCR reagents.
  3. If you work with strip of tubes, pay attention to caps - always put them in "right" order and remove them carefully not to cross-contaminate the samples.
  4. Check to see if you get the same results using alternative primers pairs.
I performed the PCR with the SAT2-Primer. It seems not to be a nice negative control. At which position do they bind?
SAT2 primers amplify genomic region of satellite DNA which represents transcriptionally inactive heterochromatin. These primers can be used as negative control for ChIP experiments with the antibody against active chromatin locus (as H3K4me3 for example). In ChIP experiments with the antibody against inactive chromatin (as H3K9me3 for example), SAT2 represents positive locus. Be sure that there is no contamination with SAT2 primers as they amplify repeated sequences that makes them very sensitive to contamination resulting in incorrect data interpretation. Unfortunately, Diagenode does not provide any primer sequences.
Using the kit LowCell# ChIP I got very high background; even with SAT2 I got the band performing end-point PCR. What can be the problem?

There are several important points:

  1. You performed end-point PCR – 45 cycles. This is too much; it is not possible to correctly quantify the intensity of bands as PCR is saturated. If you want to continue with end-point PCR, you should try to find semi-quantitative conditions by decreasing the number of amplification cycles. It is better to use qPCR for the analyse of ChIP results.
  2. Be aware that there is not cross-contamination between samples: pay attention working with strips and separate caps always putting the caps to corresponding tubes. If possible, work with barrier tips.
  3. SAT2 primers amplify repeated genomic sequences that make them very sensitive for contamination. Even small traces of human DNA are enough to provoke this contamination. DNA free barrier tips, sterile hood and gloves are recommended for working with this primers. We are aware of this problem that’s why we are looking for a new control which is not based on repeated sequences.
  4. Magnetic beads are known to get less background comparing to sepharose beads that’s why they were chosen for this kit. Note anyway that background may vary on cell type used. Moreover, this kit was mostly validated on cultured cells and not on tissues. It is possible that the tissue you use for the assay give more background with these beads.
  5. Verify that you perform properly the following steps: Keep the antibody binding beads and DNA purifying slurry in suspension while adding to tubes. Check by eye that equal pellets of beads and slurry are present in each tube. Washes (step 5) are critical.
We are looking for genes that have H3K9m3 modification in neural tissue in mouse. What control primers can we used to ensure the kit is working properly in mouse?
Mouse specific primers for TSH2B are available (cat # pp-1042-050) although we never tried these primers in neural tissue. Alternatively, your customer might take a look at the ChIP-seq results published by Mikkelsen et al. (Nature 448, 553, 2007). They did ChIP-seq with H3K9me3 in neuronal progenitor cells and these data are available from their website (http://www.broadinstitute.org/science/projects/epigenomics/chip-seq-data).
What can be used as a negative and positive PCR control?

Negative PCR controls: PCR with DNA from samples IP’d with non-immune antibodies (negative IgG). Alternatively, PCR using DNA from ChIP samples and primers specific for a DNA region to which, your antigen of interest is not binding.

Positive PCR control: PCR using input DNA.


Purification

When boiling the magnetic beads with the DNA purifying slurry, should I expect that the transparent beads in DNA purifying slurry will be at the base of the tube in suspension and will not be in suspension with the brown magnetic beads?

This is expected. Please note that in Step 6 of the protocol:

  • Pipet up-and-down before aliquoting into each tube and during the transfer of the ChIP material into the clean 1.5 ml tube (point 36).
  • Lock the tubes with tube claps (microcentrifuge tube holders) and invert them before boiling (point 38).
  • Vortex for 2 seconds at medium power (point 46).
After ChIP or MeDIP using magnetic beads, is there any step where I can store the DNA bound to the magnetic beads (before DNA purification with DNA purification module)?
Yes, knowing that it is not optimal condition that can result in DNA lost. DNA bound to the beads can be stored in last washing buffer.

Post-immunoprecipitation yields

What yield of immunoprecipitated DNA can I expect with the kit?
The results of our studies show that for the histones we can expect between 10-20 ng of DNA from 1mln cells. However the yield will vary depending on starting number of cells, antibody quality, the target - transcription factor or histone modification, and the quality of the starting sample material. Please also note that the amount of DNA that is immunoprecipitated does not necessarily indicate the percentage of specific to non-specific DNA.
What are the reasons for poor, low, or inconsistent DNA yields on a NanoDrop after immunoprecipitation?
  • Using the Nanodrop Diagenode has found that at low DNA concentration (less then 10 ng/µl) the values are not correct. The measurement at these concentrations is at the limit of detection and it results in inaccurate and inconsistent values.
  • Note that DNA purified with the slurry still contains peptides resulting from proteinase K digestion, causing inaccurate NanoDrop readings.
  • Keep in mind that the DNA is mostly in single-stranded form after boiling, so choose the corresponding option when taking the OD on the Nanodrop.
Trying the kit with two different amounts of cells I got higher recovery with lower amount of cells. Is it what should be expected?
Our in house tests often show better recovery with lower amount cells like in your experiments. It is probably due to reducing the background by lowering the Input chromatin.

Questions regarding general applications: microarrays, sequencing, PCR

How much of immunoprecipitated DNA we can get using the LowCell# ChIP kit? What is the minimal quantity of DNA per sample needed for sequencing (solexa)?

We did not test the amount of isolated DNA after ChIP using different amounts of cells. This will depend on the antibody quality, specific histone modification /transcription factor tested as well as sample used. Moreover, the amount of DNA that is immunoprecipitated, is an indication but does not give you a clear idea of the % of specifically immunoprecipitated material anyway. We can expect between 10-20ng from 1mln cells for histones.

Note that if you go for genome-wide analysis after IP, and not targeted PCR, you might need to avoid the DNA purifying slurry. A more “traditional” DNA purification can be performed using the DNA purification Module (mc-magme-002) and Phenol / Chloroform /Isoamyl alcohol (PCIA) or columns (e.g. QIAquick). These purification methods provide you with double-stranded DNA ready for amplification, microarray and sequencing.

In addition, if you go for genome-wide analysis after ChIP, you need to work with more cells (when possible). As you know, the kit is flexible depending on your sample and your application after ChIP. Up to 1 million cells can be used per IP.

Concerning the amount of DNA needed for sequencing, 10ng is sufficient. This amount you should get from 1IP; if starting with less cells for immunoprecipitation, you may need to pool DNA from several immunoprecipitation reactions. From the 10 ng or more, you go to pre-Solexa blunting, tagging, adapters binding, amplification steps.

What is the minimal quantity of DNA needed for microarrays?

1-2 µg per array has been shown to work well. To get this amount of DNA it is possible:

  • to pool several samples obtained after ChIP
  • to amplify immunoprecipitated DNA. For this application we recommend the TLDA method, after one successful round of TLDA amplification, you can provide up to 4 µg of DNA.
I am interested in using the LowCell# Chip kit to do immuniprecipitations in a non-model organism. Reading through your protocol, I was unclear on how this kit handles discovery of NEW binding sites. That is, how do you process the result DNA fragments for sequencing without knowing ahead of time what the sequence will be? Can I clone straight from the kit DNA products? Or do I need to have gene-specific primers ahead of time?

Immunoprecipitated DNA can be analysed on different ways:

  1. qPCR for known targets (you will need specific primer sets)
  2. ChIP-chip or ChIP-seq experiments to identify all binding sites. ChIP-chip experiments require additional DNA amplification as 1-2 µg are used for the hybridisation. For ChIP-seq, depending on target antibody, amplification might be skipped (if the yield is more than 30 ng).
How many cycles are required for a qPCR of the immunoprecipitated DNA? If I started with a lower number of cells (par example 1,000 cells), should the number of cycles be modified?
Normally, 40 cycles is enough to obtain the Ct value that will enable you to calculate the percentage of input DNA. However, if your experiment focuses on rare transcription factors, you may consider increasing to 45 cycles.
What are the reasons that my PCR reaction would fail (no detectable bands on a gel) even if it was successful before?

Some reasons why this may happen:

  • Using different cell types
  • Type of antibodies, primers, or reagents used

To resolve the issue of a problematic PCR, make sure you perform a PCR on genomic DNA as positive control in addition to the input and experimental ChIP samples. Remember to use the same components (Taq, buffers, primers, etc) that you use to amplify your ChIP sample. If the genomic DNA sample does not amplify, this indicates PCR failure.

I performed the qPCR according to your manual with ChIP'd sample and Input diluted 1/100. Secondly, I did make a dilution of the Input DNA (1:100, 1:50, 1:25, 1:10, 1:1) to determine the "real CT of my Input” getting the results completely different. Why there is such a difference for pol II (10.000 cells/per IP (52,48% of Input) versus 1000 cells per IP/(25% of Input)? What is the best way for ChIP results analysis? What does % of Input exactly mean?
The best is to run PCR on your ChIP'd sample and Input diluted 1/100. Do not run PCR with no diluted Input. The factor 6.66 is used to correct the dilution of Input (see manual). % of Input corresponds to % of DNA that was immunoprecipitated comparing to Input. Note that ChIP assay is a tedious method with many steps often resulting in assay-to-assay variations. The protocol should by "standardized" as much as possible (same amount of cells, antibodies concentration, etc) in order to get reproducible results. If one of the parameter is changed in the protocol (as in your case, amount of cell, resulting in different ratio Ab/beads/chromatin), the quantative comparison is quite difficult. You should define "the best conditions" for your ChIP and compare data from equivalents assays.
Should I make any modifications to the immunoprecipitation if I am doing a genome-wide analysis versus a targeted PCR analysis?

Yes, in the step: DNA isolation you should avoid DNA purifying slurry. DNA purifying slurry provides you with single-stranded DNA suitable for qPCR analysis. A more “traditional” DNA purification can be performed using the DNA purification Module (mc-magme-002) and Phenol / Chloroform /Isoamyl alcohol (PCIA) or columns (e.g. QIAquick). These purification methods provide you with double-stranded DNA ready for amplification, microarray and sequencing.

Secondly, for genome-wide analysis after ChIP, you should start the ChIP assay with a larger amount of cells, when possible.


Amplification of immunoprecipitated DNA

What amplification method, if required, is recommended after the immunoprecipitation?
We recommend T7-based Linear Amplification of DNA (TLAD). TLAD begins with a terminal transferase (TdT) tailing of the template DNA, is followed by annealing with a primer adapter containing the T7 promoter sequence. A second strand is synthesised with DNA Polymerase I Klenow fragment, and then in vitro transcription of the template generates a RNA amplification product (Liu et al, 2003). This results in linear rather than exponential amplification, and this method has the potential to reduce potential bias introduced by PCR-based amplification.
What yields can I expect after amplification?
After one successful TLDA (in general, one round) you may get up to 4 µg of DNA.
How do I generally conduct a T7-based Linear Amplification of DNA (TLDA)?
  • Start with either ChIP or MeDIP samples. You need around 10ng of DNA per amplification reaction.
  • If you do not have enough DNA pool your IPs to start TLDA.

The amplification typically yields 500 ng/µl (8 µl) of DNA, for a total of 4 µg of DNA. Microarrays usually require 2 µg per microarray.