Chromatin - Science topic

Good morning colleagues,

I want to share with you my results, hoping that someone could help me to find a possible explanation.

In the picture you can notice a 1% agarose gel where I run PCR amplicons of a gene promoter. In detail, I started from a best prognosis (left) and a worst prognosis (right) primary cell line, I fixed them with formaldehyde, lysed, extracted the DNA and performed chromatin enzymatic shearing with micrococcal nuclease; then I immunoprecipitated it with histone marks indicated in the lower part of the picture. Ultimately, I designed PCR primers to detect the presence of the amplicon in the immunoprecipitated chromatin.

As you can notice, I can observe the amplicon bands (almost 250bp), but in some histone marks IP I can also see higher bp bands which are difficult to interpretate. I exclude an aspecific amplification, as it doesn't happen in all IP bands and the primers I used should be specific for my region of interest.

Do you have any suggestion or interpretation? Have you ever encountered such a problem in your experience?

Thank you in advance

Jacopo

Dear colleagues,

I am facing a technical problem after chromatin fixation and shearing. In particular, it seems that the excess of formaldehyde used for fixation is inhibiting the RNAse, thus, my chromatin sample, which appears properly sheared on gel, presents a strong RNA band.

Have you ever experienced something similar? Do you have any suggestion to get rid of this formaldehyde excess?

Thank you in advance

Jacopo Biotti

During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated.

I've generated separate sgRNA-AAVs & Cas9-AAVs and co-transduced neurons. I've looked at DNA editing and protein, but I've gotten the occasional odd result when looking at mRNA expression. While most of my targets show reduced transcript expression following virus treatment compared to control, there are a few genes for which mRNA is undetected in control neurons but detected in the edited neurons. I can't explain this, and I know I didn't mix samples. Is it possible the gRNA-Cas9 complex destabilizes the DNA, perhaps loosening the closed chromatin state so that transcription machinery is less hindered, as opposed to control where the chromatin state wouldn't be altered? Or if not, any thoughts? My internal control (Gapdh) is consistent for all samples & treatment.

I'm doing a 3C analysis shown in the picture linked and attached below with the Bait fragments labeled and the target fragments that will be tested for interaction with the bait labeled with numbers. The picture shows two possible 3c maps based on two different restriction enzyme digests of the same region.

A, B, C, and D are regions we are considering for analysis in the future to determine if they interact with each other and with the target fragments.

Assuming we can find restriction enzymes that can digest and separate the regions shown is it possible to test between the fragments as shown for interaction or are some of them too small/too close together?

I am planning to purify the mononucleosome for cryoEM study. I have seen, people assemble the nucleosome from recombinantly expressed histones and then providing DNA sequence to it. Isn't this possible to islolate nucleosome and after enzymatic cleavage, followed by SEC chromatography for the cryoEM study ? I understand that there could be a population of others but it could be more relevant. Please suggest.

Additionally, we aim to predict whether the chromatin is in a condensed or relaxed state!!

I have recently initiated my ChIP assay and am currently in the process of optimizing the conditions for chromatin shearing using sonication. The extraction is from chickpea leaves for a total of 3 grams divided into 3 falcons.

For nuclei isolation, I employed a 25 ml buffer composition, as follows: 0.25 M sucrose, 15 mM PIPES (pH 6.8), 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Triton X-100, 1 mM PMSF, and 2.5 tablets of protease inhibitor (PI).

Additionally, for nuclei lysis, I used 2 ml of a buffer consisting of 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% SDS, 0.1% sodium deoxycholate, and 1% Triton X-100, along with half a tablet of PI.

I then performed the shearing process using two types of sonicators, and I have attached a picture that provides all the pertinent details about the sonication.

Before the gel loading I centrifuged to pellet debris, and collect the supernatant.

I loaded 5ul of sample

I am currently facing an issue with material presence on the wells and the observation of a smear or very faint band in the non-sheared sample. I would greatly appreciate any suggestions or guidance you can offer regarding this matter.

Thank you in advance for your assistance.

Could someone please suggest a protocol for DNA isolation for ChIP-qPCR? Additionally, between the PCI and Chelax 100 methods, which one is preferable for isolating DNA from formaldehyde-fixed chromatin?

Hey all,

I performed Nuclear protein extract isolation (as well as chromatin bound protein) and diluted it too much. Is there anyway I can concentrate my nuclear protein extract again?

Hello, I am using the conrad chromatin-associated RNA extraction protocol.

Everything was fine, until it was time to resuspend the pellet corresponding to the chromatin fraction, since a kind of jelly was formed, which absorbed the water.

I need to know how to solve that and if it is normal.

How can I solubilize the pellet?

I am doing a ChIP-seq with a polyclonal antibody. In order to cut down on non-specific background, I am doing a pre-clear on my samples after sonication. Basically, I rotate the samples in a +4 fridge with unblocked Dynabeads for an hour or two, separate the samples from the beads, and add the antibody.

My question is whether or not I should take out my Input Control before or after the pre-clear. I've heard of labs doing either one. Since the Input Control is used to normalize your signal to the non-biased DNA, I feel like it could go either way. If I take it before the pre-clear I would be normalizing to the total unbiased DNA collected from my samples. However, if I take it after the pre-clear, then I'm normalizing to the unbiased DNA that my antibody is actually collecting the chromatin from. Does anyone have any advice here?

Thank you for your time,

Jennifer Cheng

I have performed many chromatin extractions, but at the end of every experiment I have a large peak at 220-240nm when measuring DNA concentration with a nanodrop. I have had some very limited success by including diethyldithiocarbamic acid (DIECA) and pvp-40 in all of my solutions, but the peak remains.  I assume that it is DNA oxidation and not polysacharides because multiple ethanol washes through a cellulose column does not eliminate this peak from the spectra.  Anyone have any suggestions

ChIRP stands for chromatin isolation by RNA purfication.

I am planning to perform ChIP analysis using MNase based method from Pierce ChIP kit. The manual that comes with the kit confused me. It says we should perform sonication (20-second pulses at 3 watts power using Sonicator 3000) to break the nuclear membrane and release chromatin after MNase digestion. Doesn't sonication further shear the DNA? I didn't see any mononucleosom or dinucleosome bands on my gel so I supposed the sonication step did something funny. Would anyone recommend an alternate way to break nuclear membrane? Thanks!

I would like to extract chromatin from frozen samples, but most of them are embedded in OCT?

Does anyone has experience with it?

Thanks a lot!

Hi,

I‘m having an issue with some ChIP experiments I have been doing with a GFP antibody and protein A dynabeads. I’m basically incubating overnight with my antibody and then using protein A dynabeads to pull down the following day. My problem is that when I switch from my binding buffer (50mM HEPES 7.5, 20 mM NaCl, 1mM EDTA, 1% Triton-X-100, 0.1% sodium deoxycholate, protease inhibs) to my first wash buffer (50mM HEPES 7.4, 140 mM NaCl, 1mM EDTA, 1% Triton-X-100, 0.01% sodium deoxycholate) the beads aggregate and clump together but only in my tagged strain and not in my untagged negative control. This persists through all of my washes (0.5M NaCl buffer and 250 mM LiCl buffer) but gets much better when I do the final wash in TE. All of my washes are done at 4 degrees and the buffers were kept on ice. This has repeated several times now.

My qPCR results don’t seem completely off but the background I’m getting at loci where my protein really shouldn’t be (euchromatic regions) is quite high. I think this is because the beads aren’t being washed adequately because of the aggregation.

The pic below shows the aggregation. left is my negative control (untagged IP), right is my tagged strain.

Has anyone else noticed anything like this with dynabeads and found a solution?

any suggestions welcome! Thanks in advance!

I am trying to decipher the effect of some compounds at the level of chromatin condensation. I have looked at relaxing chromatin with HDAC inhibitors (eg TSA, VPA), which has proven to work very nicely. However, I am struggling to find any drugs that can produce the opposite effect. Any ideas on what to use?

I have looked into HDAC activators, but there are very few and generally work for a specific subtype of HDAC.

Many thanks!

Hello everyone,

In the way of this question, I would like to know if someone has anyone experienced the following:

I made a ChIP-qPCR for H3K4me3 using it as positive control GADPH promoter. For my input the Ct (H3K4me3) result mean is 27.72 and for the immunoprecipitated (IP), the CT (H3K4me3) result mean is 25.93. For another PTMs histone, my results are normal, i.e. input CT is lower than IP Ct. I would to know if someone knows if these results can affect a ChIP-seq assay.

By the way, the input Ct for IgG is 27.27 and the IP Ct for IgG is 35.79.

Thanks, everyone,

Monse

Hi I'm optimizing sonication condition in mouse primary CD4 T cells and Jurkat cells.

I tried with several conditions (10s ~ 30s ON/30s OFF, 10~30cycles) and the problem is, even I increase the number of sonication cycle, smear bands around 1kb~3kb do not disappear. Also, I can't get a fragments of 5~600bp size but the sheard chromatin bands seem to be around 200bp(First lane of the gel electrophoresis photo). I want to sonicate all unsheared big size chromatins and get fragments around 5~600bp. Also, as you can see from the gel electrophoresis photo, there's some gap between upper band(over 500bp size) and lower band(under 300bp) at right lane, and I don't know why this happens. What can I do to optimize my condition?

Briefly, my ChIP sampling protocol is like this

-Add formaldehyde(final conc. 1%) to cells in media, incubate 10min, RT

-Add glycine(final conc. 0.125M), incubate 5min, RT

-Centrifuge at 4℃

-Wash with cold PBS, centrifuge, discard supernatant (repeat 2 times)

- Lysis with 1ml SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris,pH7.5) per 2 x10^7 cells

-Aliquot lysates 300ul per IP tube --> sonication

-Centrifuge sonicated sample

-Transfer 10ul from supernatant, mix with loading dye, boil for 3 min, check the fragments by gel electrophoresis(2% agarose gel)

Actually I count my cells before starting experiment, but I did not fixed the cell concentration, and added formaldehyde and glycine dependent on the volume of media with cells. Would the cell concentration before crosslinking critical? If it does, what would be the best concentration? (It would differ by cell types, but if you have some experience with doing this, I want to know how you performed it.)

Hello everyone,

I have a small query on ChIP that I have extracted chromatin and performed sonication to obtain 200-500 bp fragments successfully from rice leaf tissue. However, I have observed an intense band with the size of ~800bp in both sheared and non-sheared (control) samples (file attached below). Can it be RNA or DNA or both? (or) Is this indication of successful or unsuccessful cross-linking? Did anyone familiar with it?

Please do the needful, and thank you for taking the time to read this. I look forward to hearing from you shortly.

I have a very non specific antibody for a protein modification and another antibody very specific for the same unmodified protein. I would like to use the antibody for the unmodified protein to pull all of it down and then use the antibody for the modified one to select for the modification of this protein. Anyone has a thought about this?

Is it possible to predict double strand breaks (DSB) (or repair??) using only ATAC data?

Can you please suggest what could be the possible reasons for the confirmation of only 4 out of 13 genes (by qPCR), not all 13? Have anyone observed the same ChIP-qPCR validation issues? Any PubMed suggestion would be great. Thank you for your help in advance.

Can anyone recommend a Myc-tag antibody that works well in ChIP or ChIP-seq for detection of a tagged TF?

We have tried to perform ChIP-seq against a Myc-tagged TF of interest [using Cell Signalling 71D10] but have not had success in achieving specific pulldown (ChIP-seq results shows ubiquitous binding across all open chromatin without any correlation to the binding motif of this TF family).

This antibody has not performed well in Western blotting either according to several colleagues who have tried (but then performance of an Ab in a Western is rarely predictive of performance in ChIP/ChIP-seq, anyway).

Any suggestions (preferably with your results attached) would be greatly appreciated so I can try another antibody with a better chance of success!

I have some mice tissues stored in RNAlater. I was wondering if it is possible to do a ChIP or chromatin accessibility assay?

Thanks in advance!

I want to use TUNEL staining to prove the association between a gene and Chromatin accessibility. However, I cannot control the experiment volume. What are the other ways to prove the association between a gene and Chromatin accessibility?

What is better to observe organelles and chromatin? Embedding cells in resin or cryo preparations for TEM?

Is there a database which quantifies or gives an idea of how chromatin accessibility or binding of a regulatory factor (TF or histone modification) is modified as a result of SNPs in the promoter or coding region. I checked in the ENCODE webpage, and it could give me the nature of the region the SNP lies in, but was unable to specifically find how the SNP could alter the same. Any leads on this would be great!

Thanks in advance!

In mammalian cell fractionation experiments, it is often observed in protocols that after removing cytoplasm and nucleoplasm proteins, you digest your chromatin with either basemuncher or MNase to fragment the chromatin, then you spin, you got the supernatant which contains some loosely bound chromatin proteins and the pellet which contains lots of the tightly bound chromatin proteins. You would need to release those tightly bound protein by resuspend the pellet with high salt buffer. My question is since the chromatin has already been digested, why not all chromatin is in the supernatant after basemuncher digestion and spin? In other words, why the tightly bound chromatin proteins are not released into supernatant in this step?

Is it possible to map chromatin interactions associated with EBV genome in EBV infected cancer patients using hic data without WGS?

Hi,

I am interested in a chromatin state over one of the promoters in different cell lines.

I would like to check it by ChIP-qPCR.

The question is how to show it? Is a one set of primers enouth and just do three repetitions? Or do you need more? If yes, should they be overlapping/close to each other or rather far away?

Also, how many chromatin markers do you need to check? Is just one fine?

Many thanks for any help!

Hi,

I want to do ChIP on jurkat cells but with my protocole I can't fragment the chromatine enough (fragment now are between 3000 and 1000 bp), even at 1h of sonication....

Do you have advice? (sonication, buffer, ...)

To charaterize TF function in specific bone marrow or immune cell subsets, we only have relatively small cell numbers (10⁶ or less) to prepare chromatin from. Does anybody have (good) experiences with high sensitivy ChIP kits or home-made protocols for small populations? Cheers and Thx 

I am looking for a sonicator with a reasonable price for our lab, which I can get consistent results in DNA chromatin shearing. I appreciate any suggestions and your experiences with chromatin DNA sonication for the Chip-seq experiment. Thanks in advance.

I have extracted total DNA from an aliquot of sheared chromatin and ran 250 ng on 1.5% agarose gel (stain with Syber safe). I have added 40% sucrose as a loading buffer. I think my sonication has worked fine but the DNA band doesn't look right. Could you please tell me if this the problem of gel or sample?

One important process of Chip is to shear the DNA into ~300-1000bp fragments using enzymatic digestion or sonication? So why should be ~300-1000bp?

I know that shorter fragments less than ~300bp may lose the DNA-protein binding sites? This may be one reason of >300bp?

But what will happen if fragments >1000bp?

I'm looking for a fixative that works quickly but you can safely store cells in for a longer period (i.e. a few hours). Most importantly it must be able to fix chromatin well and be compatible with fluorescence in situ hybridization (for chromosome labeling).

I have read about RNAlater being able to let cells/tissue sit in it for a while, but I am not sure if it would interfere with the chromatin or FISH experiment.

Any advice is appreciated, bonus if you can provide sources. Thanks!

We are trying to design an experiment investigating chromatin co-localization of two DNA binding proteins in the system where both endogenous genes are knock-out and re-expressed with attached tags. Most of published Re-ChIP experiments that I came across utilize one anti-tag antibody and second protein-specific. That makes me wonder if there are some technical or conceptual disadvantages of using two tags in this type of experiments. Thank you in advance for all your input.

Hello all,

I want to ask if you can proceed with under-sonicated chromatin by thawing on ice and sonicating extra cycles, will affect/compromise the chromatin quality laters with IP? I am doing double fixation protocol (DSG + FA) and sonicate using Bioruptor with 0.1% SDS final Conc. lysis buffer.

Dear All, I want to preform ChIP-seq experiment on a transcription factor and Methyl-seq for analysis of methylation patterns

are there any tips that I should consider to prepare samples?

Tips and suggestions are most welcomed

Hi everybody,

I am doing CHIP experiment. The problem is that in my gel agarose I see alot of DNA with big size (longer that 1000 bp) which means there is alot of unfragmented DNA in my gel.

I increased the enzyme amount but still I see alot of unfragmented DNA in my gel agarose.

Can someone help me how can I increase the chromatin digestion in my sample?

Thanks in advance

Elmira

What are the benefits of Sonication vs Enzymatic digestion (or vice versa) for ChIP and ChIP seq?

We are planning to send Prostate cancer cell line samples for ChIP-seq, and getting chromatin fragments ~500bp after sonication (we do not have the water bath sonicator). The Core that we are sending the samples suggested we use enzymatic digestion to get proper chromatin fragmentation (100-300bp) for better results.

I am currently using a bench top Sonicater at 30% amplitude for 20 cycles (15 sec on, 45 sec off). I am getting chromatin fragments around ~500bp which works for our ChIP-qPCR, but despite trying optimization, we are not getting fragments in the range of 100-300bp, that is required for ChIP-Seq.

What protocol do you use and how do you get the proper fragmentation of chromatin in your ChIP and ChIP-Seq experiments?

Hi,

I am planning to test a drug that is known to reduce phosphorylation of RNA poll II at Ser2.

My question is when I collect lysate for western do I need to do nuclear fractionation or is whole cell lysate sufficient?

This is because I read that chromatin bound proteins might be hard to detect from whole cell lysates but I do see westerns of RNA pol II from whole cell lysate on images of representative western blots on the antibody webpages online.

Thanks

Hi everyone,

In order to do CHIP experiment, I used some lysis buffer and micrococcus followed by sonication to digest chromatin of the cells. I did all the process of cross-lincking, quenching, and cell lysis and chromatin digestion and then DNA purification and then I loaded the DNA in gel agarose but I see a lot of smear in longer fragments. Can someone guide me how can I know that these smear belong to the un-lysis cells or un-fragmented chromatin. how can I be sure that they are not chromatin. I uploaded the gel image and these huge smear in the large part of the gel is visible.

Hi everybody,

I wanna use an antibody to determine the presence of a protein in my chip-seq experiment. Can someone help me how can do concentration optimization for antibody which I use ?

Thanks in advance :)

Hello, everyone!

I'm going to start a project with single-cell ATAC-seq for analysis of open chromatin regions from freeze tissue samples.

I'm lost about the nuclei concentration.

If I made a nuclei isolation protocol from the human brain freeze tissue sample (hippocampus), what would be the nuclei mean number from these?

2000 nuclei? 5000 nuclei?

There is any paper about that?

Thank you!

I am working on epigenetic modifications with aging, especially DNA methylation. I have .bed, .sam and .bedgraph files obtained in bisulfite DNA sequencing.

1) Is there any software other than methylkit for DNA methylation analysis? I am looking for global and chromatin level DNA methylation analysis.

2) Any tool that can analyse CpG methylation level in specif regions e.g promoter region, 5 and 3 UTRs or a specific genes.

Hi all,

I am performing a Co-IP experiment where would like to check the binding of my protein to the DNA of interest. I kept a control where I incubated the DNA and antibody only with the protein A/G beads, after which I performed PCR to amplify the DNA of my interest.

I am seeing the desired PCR bands even in the control. Is it possible that the protein A/G beads show some sort of nonspecific binding to my DNA of interest resulting in these bands? I found the following links mentioning nonspecific binding of DNA to Co-IP beads:

Any help is appreciated!

Thanks!

Specifically, I would like to know how histone modifications that are associated with changes in gene expression, change the flexibility of the chromatin fiber. For an example, does H3K27ac make the chromatin more flexible, and by how much?

Specific references would be appreciated because I can not find them.

The most difficult scientific advances become possible when you ask the right question. In the case of COVID-19, I understand that the right question is: why do some people become seriously infected when exposed to low viral load, while others are not sick even when exposed to high viral loads? The situation resembles the film The Andromeda Strain, which I used for many years as an example of scientific research in my Philosophy of Science classes. There is a deathly virus that came in a spaceship, which kills the vast majority of people. However, a baby and a drunk are immune to the action of this virus. A scientist then asks the right question: why are these two people immune? When investigating the physiology of these people, he discovers that both have their PH in certain ranges, the baby alkaline due to excessive crying, and the drunk acid from the ingested drink. In the case of Corona, we have indicators that its action occurs mainly in the mitochondria (see Castalia Francon´s insights below), which has an evolutionary origin due to endosymbiosis (when the eukaryotic cell incorporates bacteria that help it perform certain functions). Mitochondrial DNA is exclusively maternal in origin. Some factors that affect the mitochondria, such as nicotine and metformin, may predispose to the action of the virus. All this indicates that it is necessary to research the affinity of the virus with certain segments of the mitochondrial DNA, to verify if there is a segment present in the mitochondrial DNA of susceptible people, which is not found in the DNA of people not susceptible to the disease. After this discovery CRISPR technology may be used succssfully.

The mechanisms of action of Metformin against Diabetes, boosting mitochondria and reducing the stress caused by excess glucose, may be useful for the fight against the new Coronavirus. I have discussed with experts the action of Metformin and its relation to cancer tumor genesis in another RG question (https://www.researchgate.net/post/Does_the_use_of_metformin_against_type_2_diabetes_increase_or_decrease_the_probability_of_appearance_of_cancer_tumors).

To answer the above question, it is necessary to take into account recent data on the mechanism of action of the virus and empirical statistical (positive or negative) correlation between use of Metformin and the lethality of the virus. Is there any data on this correlation?

According to comments by Castalia Francon, "Neutrophils are known as the core of the 'innate immune systems". They are our 'first responders" to any imminent medical holocaust...NETS, 'Neutrophilic Extracellular Traps" as the battlefield. These powerful products of our neutrophils are essentially a nuclear option against the pathogens. As such they do whatever they can to prevent that explosive death of the neutrophil that will ensnare them...

For bacteria and viruses, their goal of survival entails first avoiding, by whatever means they have, the phagocytosis efforts and then countering the next move of the neutrophils, their death by NETosis.

To attempt this, they seek to manipulate the communications between mitochondria and the cell so as to stall the Neutrophil in a limbo known as "autophagy" while the microbe can continue to reproduce and spread.

The term "autophagy" came into use to denote a systematic, almost "thoughtful" process by means of which the neutrophils, just like macrophages, could move sequentially through a series of considerations pertaining to how to deal with their situation in terms of life and death. But by its very nature as an extremely complex process entailing many dimensional considerations, it is also readily exploitable by pathogens, and in particular by viruses

Many pathogens have been shown to evade or exploit autophagy in macrophages, aiming to establish an intracellular niche for long-term survival and replication Subversion of autophagy by microbes in neutrophils is far less studied but the Corona situation seems to point to it...Our best analogy to it is how when we with to delete", 'our computers ask us, "Are you sure'?" It appears most likely that autophagy is the manifestation of a process that almost always goes on, and eventuates in some adaptive response....unless, of course, the communications between mitochondria and cell are distorted or diminished by pathogen actions.

Viruses and bacteria have evolved over millions of years as each others nemeses, long before we were on the scene, thus the virus is naturally inclined to maniupulate the mitochrondira and by means of that to in turn manipulate the entire cell which is driven by the mitochondria and their needs...

Unfortunately the current 'expert' narrative speaks dichotomously of either "promoting" or 'inhibiting" autophagy instead of coming to terms that there is an ongoing and complex battle going on where any one aspect can shift the nature of the complex decision making.one way or the other. The pathogen, however, is most happy when no clear decisions at all is reached.

The essential treatment for Corona Virus infection is to assure that once the phagocytosis fails....which it often does...that the neutrophils are not stalled from their productive and special death with its Neutrophilic Extracellular Trap creation...which releases the remarkable power of their own DNA to act as a potent immune system weapon against microbes.

We would not be surprised if the CRISPR technology which is now surfacing with so much promise is not an aspect of the manner in which DNA/chromatin sections can be used productively to kill viruses...as they have been used for millennia, apparently, by bacteria.

In summary

• Previous smoking habit is highly associated with the dire effects of Corona Infection....and it also leads to a maladaptive tendency towards' "autophagy" which renders those persons' neutrophils unable to engage in "NETosis " and create "NETS""t

• Smokers neutrophils seem to have phenotypically altered over the course of their previous smoking habit so that they are unable to properly engage in their intense first line of defense, phagocytosis (via NADPH mechanisms).

We only need to observe that in Italy, where the death rates from Corona are soaring, they differ in the terms of their 'lockdown" from ours. Whereas, in the US, pharmacies and groceries and other vital retailers are excluded, in Italy, stunningly, tobacconists and vapes shops join pharmacies as essential and are exempted from lockdown.

Normally, the generation of the ROS products via phagocytosis is a prerequisite for the subsequent death via NETosis.. This is unsurprising since that a failure to be able to engage in phagocytosis would already connote some abnormality or deficit.

•It has however been found that even when the initial phagocytosis does not occur, that, if system can be induced downstream to generate ROS, then NETosis and the creation of those 'traps' is still possible.

• Chloroquine is known to be an "autophagy preventer" in that it can serve to rescue the neutrophil system and thus precipitate the creation of 'NETS. It seems to achieve a mobilization of the NET trap system even though phagocytosis has not succeeded. Exactly what it does is still not clear.

It appears that it is optimal to administer chloroquine early, but only after the first phagocytosis efforts of the neutrophil system but before the virus has seized the opportunity, by manipulating the communications between mitochondria and the cell, to spread and do maximum damage".

Nuclear pellet was resuspended and incubated in nuclear lysis buffer and then centrifuged to load the S/N.

I am using the Covaris S220 with a 1ml Glass Tube Cat#520056 to shear crosslinked yeast chromatin. I have a lot to optimize going into this protocol with a new strain but found only one source that recommended a range of 0.5-1.5e7 cells/ml for mammalian cells.

Is this a good place to start? Do you have a better recommendation for yeast?

PS:

Currently my setting are as follows if that helps.

1ml, PIP:240W, DF:20%, CPB:200, 4C

I am trying to identify a kit to assay glucocorticoid receptors (GRs) bound to GREs in a select brain region. I have an antibody to perform the immunoprecipitation part of the assay, but I am trying to identify a kit. This is a kit that seems to work for some people.

https://www.epigentek.com/catalog/epiquik-chromatin-immunoprecipitation-chip-kit-p-59.html

Any suggestions?

I've just started my ChIP assay and am currently optimizing the conditions for shearing of chromatin by sonication using bioruptor sonic . Sonication was performed under these conditions: first group 30 seconds ON, 45 seconds OFF, and35 seconds ON, 45 seconds OFF for various cycles15, 9 cycles subsequently, low high botton on HIGH. Reverse cross-linking was performed by aliquote15ul of the sonicated sample to 180 ul of de cross-linking buffer (DCL1). then incubation for 10 min at 37c, then 4ul of RNase incubate for 10min at 50c followed by 65c /50 min. My aim is to shear the chromatin to around 100-500bp. Attached is the outcome . can someone help me in the optimization of the sonication? each time I check the fragment size using a tape station I didn't get a good result? also when i precipitate the DNA i didn't got any good result ?any idea

thanks

hello everyone, please, could someone share with me a protocol for chromatin immunoprecipation (ChiP) assay with monocytes? With my best regards. Marie-Yolande

Hi..

I'm wondering if anyone can recommend a protocol to extract chromatin from 3D spheroid cells. I have glioblastoma patient derived (GBM) cell line that turns into spheroid and I want to extract chromatin for ChIP-seq. I know the general protocol for chromatin extraction from adherent cells but I would like to know if there is a specific protocol to deal with spheroid cells. Any suggestions?

Hello,

We're currently trying to use the MAGnify ChIP kit for Chromatin immunoprecipitation with HeLa and LNCap cells, but we're having some issues, when it comes to Chromatin Shearing.

In the kit's protocol they give instructions on the Chromatin Shearing using a Covaris S2 sonicator:

Duty Cycle: 5%

Cycles: 10

Intensity: 2

Temperature (bath): 4°C

Cycles per Burst: 200

Power mode: Frequency Sweeping

Cycle Time: 60 seconds

Degassing mode: Continuous

However, we have a Covaris E220 evolution sonicator instead and many of these options seem to be unavailable; Instead of Duty Cycle it has Duty Factor (though, i assume it is the same option, simply renamed), but the Intensity option is replaced with Peak Incident Power in Watts and it doesn't seem to have the option to choose the amount of cycles and their cycle time, instead having a single option - Treatment time.

So i'd like to know if there's a way to translate the different options between the sonicators, or if anyone has experience using Covaris E220 for Chromatin Shearing to share their methods.

We're using 1 million cells in a 50 μl sample volume of MAGnify ChIP kit's lysis buffer.

Any help would be appreciated.

Hi, I am currently trying to optimize a Chromosome Conformation Capture (3C) experiment to determine physical interactions between two cis-regulatory modules (CRMs). My main concern is that the CRMs are located approximately 5 kb apart in the genome.

My model organism is the purple sea urchin (embryos). I fix, isolate nuclei, and digest them with BglII RE and subsequently ligate them. To determine interaction, I am using semi-quantitative PCR. Basically, I have an anchor forward primer and test forward primers of varying distance (all in the same sense direction). And for each of the primers, I have a corresponding reverse primer to determine primer efficiency.

I am wondering if a simple 3C experiment will be able to provide good evidence of interactions at the resolution of 5 kb?

I would appreciate any input! Thank you!

I want to increase chromatin compaction of cancer cell and to see the function of interested gene in this process. Is there any method to make it?

Gene expression is important for proper functioning of particular cell type. It is regulated by set of transcription factors (TFs) in a specific chromatin environment. It is possibe to predict gene expression alone by TF or chromatin marks? I want to know what is the correct hierachy of such organization and which one interact to whom or there is a kind of feedback mechanism between such features.

Thanks.

Hello, I want to perform a Chromatin Immunoprecipitation Protocol (ChIRP) and I am gone to rely in Chu,2011 Mol.Cell (https://www.ncbi.nlm.nih.gov/pubmed/21963238).

In this protocol they use Dynabeads® MyOne™ Streptavidin C1. Can I use other type of streptavidin beads for this experiment? I really want to know it because dyneabeads are expensive and I am planning to perform a lot of precipitations.

I have seen other cheaper types of streptavidin magnetic beads from different brands (like MagnaBind™ Streptavidin Beads).

Thank you.

Diego Gómez

Cre recombination efficiency often impacts activation of Cre/LoxP dependent transgenes. We have recently encountered problems with recombination in certain cell types in vivo, especially with inducible recombinase (CreERT2). The recombination rates range from very high (>90%) to barely detectable. Increasing Cre concentration/action time by prolonging TAM induction regimens seems to improve it, albeit very modestly. Our transgene is in the Rosa26 locus, which many regard as a safe harbor. Interestingly, the same Cre recombinases (Cx43-CreERT2, Calb2-CreERT2, were amongst the problematic ones) seem to perform much better in other loci. We are considering two possibilities:

- methylation of the gene - this may effect recombination (e.g., if LoxP sites are methylated), but may be also happening independently

- closed chromatin leading to reduced Cre access to the locus and/or reduced overall expression of the gene

In any case, I am toying with the idea of using an alternative STOP cassette, flanked by 3 LoxP sites on each side, to increase the efficacy of recombination. It may not help if the issue is not cre related, but I don't see any risk with this approach. A slightly higher chance for random recombination seems a small price to pay if using such LoxP triplets (all facing in the same direction) would help. I would appreciate feedback from someone more experienced, about pros and cons of this solution.

On a related note: I am curious about the relative efficiencies of Cre, Flp and PhiC31 on different target sequences (e.g., LoxP vs Lox2272, AttP-CT/AttB-CT vs AttP-TC/AttB-TC, etc.). I would especially interested in strategies to optimize Cre/LoxP and Flp/FRT system, but I am also considering PhiC31 for the same purpose (especially that the latter works in unidirectional way - I see this and an advantage - and this paper shows that PhiC31 may be superior to Flp). I am aware of the one report discussing PhiC31 causing (potential) DNA damage, but I would love to hear more opinions. Regrettably, the literature mentions the above mentioned issues are very sparse; I failed to find any relevant recent papers. I would be delighted to hear expert opinions of fellow researchers.

I need a method to evaluate chromatin of rat sperm.

I did chromatin extraction using both commercial kit and homemade recipe. By using RNase A and protease K digestion together with phenol-chloroform-isoamyl or on-column cleaning, I then isolated the sheared DNA directly after chromatin extraction prior to the proceeding to immunoprecipitation so as to determine the shear efficiency and quantify DNA using High sensitivity DNA chips from Agilent. From the results, my DNA concentrations were hundreds of pg/uL, but I do not know how to use that to calculate the amount of chromatin.

And the second question is, since I work with chicken samples, should my positive control for ChIP (anti-RNA polymerase II) be specific to chickens, or since RNAP II is highly conserved in eukaryotes, general ChIP-grade Ab should be fine? And for the controls for IP (I used RNAP II and non-immune IgG), should they also be used as the positive and negative controls in the qPCR later, or the qPCR should use genomic DNA as a positive control and use input DNA reverse cross-linked directly from chromatin extraction as reference? And in such ChIP-PCR study, should the reference genes also be used for final calculation?

For the analysis, t is indicated in many protocols that one should use either the percent of input DNA, which is reverse cross-linked from chromatin extraction, or fold enrichment in according to the negative control (e.g. non-immune IgG). However, since the non-immune IgG should not have reactivity to any specific loci, but only provides the information how much background is presented in the product, shouldn't the Ct value be extremely high or even undetermined eventually? If it is undetermined, how could I calculate the fold enrichment then?

Thank you for your time!

I have seen lots of papers in literature using prep cell for purifying nucleosomes (from DNA and hexasome species), but I couldn't seem to find another protocol apart from that discussed by Luger et al in 1999.

I was wondering what is the typical yield from the protocol? How low can the protein sample input be to get recovery? My input sample is relatively small (1-2 micrograms), so I'm afraid I won't get any recovery. I have been using low volume density gradient centrifugation to separate nucleosome and DNA with mixed success, and was wondering if the purification resolution can be improved using prep cell

I normally use this protocol for ChIPseq:

After chromatin sonication, it says to add 1/10 volume of Triton X 10% to the samples, spin to remove pellet debris and then incubate beads/chromatin o/n.

What does the triton do in this step?

Thanks

My lab is performing ATAC-seq on D. Melanogaster brains. In many steps, we centrifuge our samples at 800 rcf, but we find that the samples struggle to pellet at this speed. If we were to increase the force of centrifugation, would we risk altering the chromatin confirmation?

Hi, I am looking to assemble a customized kit for ChIP-Seq of low abundance proteins. Can anyone recommend sources of solid phase support that will reliably recover antibodies (columns/beads coated with either protein G or streptavidin) while having low affinity to DNA and chromatin?

Hi,

Based on results from ChIP-Seq experiments I chose ~10-15 potential chromatin interactions in different loci in the genome which I want to check.

Does anyone a suggestion which approach will be the best to check them? Should I choose a method which is followed by PCR or sequencinq, which one will be better time- and cost-wise to check these 10-15 interactions?

Thank you for any help!