ribo-seq分析流程

hi， my name is ellen wines， apple and i'm a product manager at epicipher this is part two of a three part series on the basics of chromaton mapping in part one， i covered the fundamentals of chromatin structure and gene regulation in part two， i'll review the basic steps of chromaton mapping data analysis and considerations for selecting a chromaton mapping assay， so let's delve into an actual chromaton mapping experiment here we will cover the basic steps of the most commonly used assay chipseak before starting you have to collect some information first， what are your samples？ these are the cells or tissues？ you want to use for mapping？ second， you have to select the targets you want to map this refers to the pissed on ptm or protein you want to map in your cells in my example， i have two samples wild type and mutant and i want to map two targets in each of them third calculate how many reactions you need to set up it is important to remember that in mapping assays， you can only map one target and one sample per reaction so for my example to map two targets and two samples， i need to set up a total of four reactions i also need to make sure i have enough of each sample to perform two reactions now let's go into the actual assay chipseak stands for chromaton amino precipitation sequencing the first step is to cross link cells， which stabilizes targets on chromatin this step is important to optimize since too much or too little fixation， negatively impacts， healed background and data quality you then lie cells and isolate chromatin the chromatin is fragmented into small mono nucleuse own pieces chromatin fragmentation is another challenging step to get right and also needs to be optimized for each cell or tissue type after fragmentation add an antibody to the targeted interest which binds nucleusiums containing your target for histone ptms as shown here it is essential to use a rigorously validated antibody since many antibodies to histone ptms show nonspecific binding activity you then use the antibody to pull down the nucleuseums associated with your target this is the amino precipitation or ip step and includes a series of stringent high salt washes to fully strip unbound nucleuseums and reduce nonspecific background unfortunately these washes can also result in loss of your target associated nucleosomes particularly for weak chrometon interactions or if the ant body has low binding affinity additional optimization may be required after pull down you digest proteins reverse cross links and purify dna then you prepare what are called sequencing libraries？ this process adds unique barcodes to each reaction so sequencing data can be assigned to the correct reaction during analysis and after library prep you're ready to sequence sequencing produces millions of short reads and these reads correspond to where your target is located across the genome scientists next align or map these reads to the matching sequence in the genome here i'm showing a small segment of the genome as an example， but sequences are mapped genome wide mapping the reads allows you to know the amount of target at each location on dna this is called enrichment areas with many aligned reads have high enrichment while areas with few reads have low enrichment to visualize data aligned reads are normalized and used to generate sequencing tracks more mapped reads or enrichment generates peaks in sequencing tracks and no enrichment means there are no peaks again tracks are generated for the whole genome we are just looking at one area here， but you can look up any gene you want and see how much of your target is bound we used chipseak as our introductory example， because it has been the standard chromaton mapping tool for many many years， but it is not the only option how do scientists select an assay for their project， it is important to think about the number of cells high cell requirements limits analysis of small samples and how many targets you can map processing time and throughput are also key steps increases the risk of sample loss makes it difficult to perform multiple reactions at one time and slows down data generation simplicity of the workflow is also essential since complicated assays lead to higher variation and more user error the major cost of chromaton mapping assays is sequencing more sequencing per reaction， increases costs and restricts experimental scale and finally you want data specific for your target with low off target background reproducibility across reactions is also crucial so how does chip seek measure up well despite its widespread use you might be surprised to hear that it fails every single one of these metrics it requires millions of cells and is not ideal for rare or precious cell types it takes approximately a week to perform an experiment and has been hard to adapt for high throughput many steps require cell or tissue specific optimization the harsh wash steps during ip can also reduce target yields chipseak requires many reads per reaction and is the main cost of the assay and even with all of these investments the resulting data are often poor quality and unreliable chip has had a dominant hold on the field for decades， not because it is an ideal technique， but because nothing else was readily available chip was robust enough， but improvements are clearly needed to address these problems epicipher offers katana cut and run and cut and tack assays for ultra sensitive chromaton profiling our assays and user friendly protocols pass each metric for assay selection inside by side comparisons with chipseak you can see that cut and run and cut and tag require fewer cells and sequencing costs katana assays are also much faster you can go from cells to sequencing in as few as two days furthermore the data are greatly improved there is less background and more robust target signal join us in part three of the chromaton mapping series to learn more about cut and run and cut and tag assays。

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