ChiP seq | Chromatin immunoprecipitation and sequencing | Methods in biology | CSIR NET

in this video we'll be talking about chromatine imuno precipitation and sequencing it's also known as chipseek so chipsi is a method to study DNA protein interaction in a genome wide scale this is a technique that can help us to understand where does a transcription Factor binds in in a genomic location it's a powerful tool to identify genome wide Association of several DNA binding prot or transcription factor or any other histones let's say so here is a situation one and two in situation one a transcription Factor marked here in red binds to a specific genomic location

so it has a very specific binding site in the genome it doesn't bind to every site or many sites in the genome it is also possible that there is another transcription factor that binds into many genomic locations whatever be the scenario these kind of questions can be answered by chipseek so let's see how this process work first of all antibody is incubated against the transcription factor x so we need an antibody against the transcription Factor whose binding we are trying to investigate so in this case this antibody detects the red transcription Factor after that there

are too much chromatin associated with these transcription factors so we need to sever the the chromatine by sonication so there are small fragments of the chromatine which are generated after sonication that you can see in Step number two in Step number three a pull down experiment is performed where on a magnetic bead these antibodies are coated and the portion of the chromatine which is bound by this transcription Factor will be pulled down because there is an interaction between the antigen and the antibody and it would be use and the Magnetic pull would be used to

pull down these fraction from the pull down fraction the DNA fragments would be recovered so these DNA fragments are bound by transcription factor x next question is where does these DNA fragment belong into the genome so these fragments would be sequenced and these is known as reads after these reads are generated they would be aligned against the existing genome and then a peak calling would be performed Peak calling simply means how many Peak for a genomic location one can find so basically if there are let's say 10 fragments in a particular mapping to a particular

genomic location that means the transcription Factor highly binds to that particular region so in this case if we see here these reads or the fragments all correspond to this particular genomic location and these genomic location can be obtained from genome assemblies in the uh databases so this peak height means house strong would be the binding so here you can see there is a strong binding here is a strong binding but there is no apparent binding in this particular region so how does the data look like data would somehow look like this so you can see

the transcription factor x chap uh chip peak in this case we can see the peak is colocalizing with a gene uh and the promoter of the gene mostly so we can conclude from this data that the particular transcription Factor binds to the promoter region of these Gene do look at the chip uh chip peaks in other genomic locations and you won't see much of that in the other places okay there are many other applications for example mapping the transcription Factor binding epigenetic modification studies histone modification profiling identification of enhancer promoters Etc so be I mean

forget forget about all these applications now let's try to understand it in bit more details with some examples in this case the specific hones are used for the chipsi experiment so antibody was used against the h2a Z variant of the histone and it was note that where does these histone variant is mapping to it maps to the promoter of this particular Gene also H 3.3 which is enriched in a promote and Gene bodies of actively transcribing Gene can show us this particular Gene marked here is active another chipsi experiment here is serin 5 phosphate of

RNA polymerase 2 so serin 5 phosphate phosphorilated RNA polymerase 2 is an active RNA polymerase so in this case if you get a lot of Chip peek all along the body that means this Gene is actively transcribed so now we are looking at different different different chip signatures and by combining all these signatures we get a detailed Outlook into the transcription process so that is the power of chipseek a it can give you an O overall idea and deeper insight about transcription also it can give you a genome wide understanding about a transcription factor or

a DNA binding protein binding now often chips are combined with many other techniques such as attack sequencing on which I have made a detailed video so click on the I button to get that video so often chipseek RN and ax should be combined together to understand the transcription Factor binding the chromatin accessibility and the gene expression so in this case let me walk you through this data neuron and fibroblast are uh kind of compared for this Gene a transcription profile so in this case the rnsc data tells us the expression of Gene a is less

in fi blast more in neurons and if we look at the chip Peaks you can see the transcription Factor one particular transcription factor is binding more in the promoter present in the neuron and it's not present that much in the fibroblast so it's basically a cell type specific transcription Factor also it can be seen that the promoter region was much more accessible in this case so atoi give us an understanding about accessibility of the region so the promoter was accessible now the promoter is bound to that transcription Factor as a result there is a gene

expression for Gene a and in the fibroblast the promoter was not that accessible transcription Factor also doesn't bind and in this case you see a very less expression so more and more layer of information about transcription is uncovered when chipstick is combined with other techniques so I hope this video was useful and we can talk more about these techniques in detailed videos stay tuned for more get more notes and flash cards in our Facebook page or Instagram page links are provided in the description you can support our Channel using PTM PayPal or UPI see you

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