COMIN Interaction analysis using powder and tag sequencing is a method for genome wide comin long range comin interaction analysis. In short, we call this magic share pad. This method involves many molecular biology steps and can be complicated.
Therefore, in this following videotape, we'll show you some of the key steps involved in com, immuno immunoprecipitation, and the library construction for the chair. PET analysis Chromatin immunoprecipitation known as chip, is the first critical step in constructing a chair pet library. Here, chromatin samples from cell cultures are cross-linked sonicated and immuno precipitated.
Separate aliquots of chip DNA are then ligated to barcoded half linkers and proximity ligation is carried out. Pets are released by restriction digestion, purified on coated magnetic beads, and li gated to adapters for next generation sequencing. Chimeric chair pets are identified via the AB linker composition and tag flanking.
The central linker sequence are read out and mapped to the genome. In this video, we will be showing the standard chip protocol routinely used in our lab on a brass cancer cell line MCF seven From our data, higher chip enrichment with specific antibodies yields better chair pet data. Hence, we strongly recommend optimization of the chip protocol and enrichment validation by QPCR.
Before proceeding to chair pet library Construction CF seven cells are cultured to about 75%co fluency in a 500 centimeter square tissue culture dish, which contains approximately 20 million cells before cross-linking. Discard growth media and wash cells twice. With warm PBS fix Cells with freshly prepared 1.5 millimolar EGS for 45 minutes on a rotator at room Temperature At 37%formaldehyde to a final concentration of 1%and incubate for 20 minutes.
Crosslinking efficiency is critical and varies with cell lines and the protein of interest. Crosslink a concentration incubation time and temperature should be optimized. Quench the cross linkers with a final concentration of 200 millimolar glycine for 10 Minutes.
Discard quenched cross linkers and washed twice with cold PBS Hover cells by scraping pallet cells with a refrigerated benchtop centrifuge, The pallet is now ready for cell lysis. Alternatively, freeze palate at minus 80 degrees Celsius. If cell palate Is frozen, thaw on ice before proceeding with cell lysis resand palate with 0.1%SDS lysis Buffer.
Rotate the tube at four degrees Celsius palate with a refrigerated centrifuge, discard Supernatant and repeat cell lysis. Once nuclear lysis is performed to release cross-linked chromatin before chromatin fragmentation. The absence of nuclear membrane allows the cross-linked chromatin to be sonicated under gentler conditions.
Reus Band nuclear palette in 1%SDS lysis Buffer. Transfer the suspension to a high speed centrifuge tube. Rotate the tube at four degrees Celsius.
Remove a nuclear debris via centrifugation. The chromatic palette should look like this. Use a P 1000 piper tip to break up the palette into pieces and wash twice with 0.1%SDS lysis Buffer.
Rotate the tube at four degrees Celsius palate with a refrigerated centrifuge. The nuclear palate Is now ready for sonication. Alternatively, freeze palate at minus 80 degrees Celsius.
Transfer palate to a clear tube Add 0.1%SDS lysis, buffer to the palate and remove all bubbles. Keep samples cold throughout sonication to prevent overheating. Different conditions such as buffer volume, probe depth, sonication strength, number of cycles and extent of cross-linking can influence sonication efficiency.
Reverse cross-link and aliquot of chromatin using proteinase K and run an aros gel. To verify fragmentation efficiency optimization is recommended to obtain the desired DNA size range between 200 to 600. Base pair remove cell dri by centrifugation.
The chromatin is now ready to be pre-cleared with magnetic beads. Alternatively stopped at minus 80 degrees Celsius until sufficient chromatin is collected. To start a chip Wash beads three times with beads wash buffer this and future washes involving magnetic protein beads consist of the following steps.
Add sonicated chromatin to the pre-washed beads and incubate overnight at four degrees Celsius in a separate tube. Coat the pre-washed beads with antibody and incubate overnight at four degrees Celsius. Wash the antibody coated beads twice with beads wash buffer.
The high Complexity of substrate for proximity ligation inevitably leads to substantial non-specific noise, thereby increasing the cost of sequencing. Apart from reducing the level of complexity and background noise. We used a mouse RNA polymerase two monoclonal antibody to identify chromatin interactions involved in transcription regulation.
This allows the investigation of active promoters and their corresponding regulatory regions, allowing the understanding of efficient and possibly cooperative transcription. Discard wash buffer from the antibody Coated beads combined Pre-cleared chromatin with antibody coated beads and incubate overnight at four degrees Celsius after overnight incubation, the beads are subjected to a series of washes to remove unbound chromatin resand beads In TE buffer aliquot. A small amount for quantitative And enrichment check chromatin interaction analysis using pad and tag sequencing is a technique developed for large scale de novo analysis of higher order chromatin structures interacting.
DNA fragments which have been enriched by chromatin immunoprecipitation are captured by proximity ligation. The pad and tag strategy is applied to the construction of CHI PET Libraries, which are sequenced by high throughput next generation sequencing technologies. In this video, we will show the critical experimental aspects in CHI PET library construction.
The first half of the library construction involves manipulating protein A or G beats, which the DNA fragments are bound to, to remove the enzymes and buffering salt. After each enzymatic reaction, use gentle centrifugation and flicking action for each washing step. Pipette the supernatant several times to maximize the capture of beads when placed on a magnetic particle.
Concentrator for all incubation steps containing beads in the reaction mixture. Prepare master mix beforehand and add enzyme last after the beads are resus resuspended homogeneously Ensure that the beads are well mixed throughout the entire incubation. Half linker oligo nucleotides are designed with an internal barcode consisting of four nucleotides and recognition site for the type two S restriction enzyme MME one.
After ligating the half linkers to the tethered DNA fragments in C chromatic complexes, the interacting DNA fragments will be connected by a complete linker sequence. These half linkers generate sequences with heterodimer ab linkers derived from non-specific ligation products or sequences with homodimer, AA or BB linkers derived from specific ligation products. Hence the use of two half linkers with different nucleotide barcodes allow specifications of different experiments or replicates as well as monitoring of the non-specific chimeric ligation rates between different chip complexes.
It is important to set up the reaction on ice And thaw the half linkers gently on ice. Combine half linkers well with water and then with a PEG containing ligase buffer. Ensure the master mix is well mixed before resus.
Suspending the chip DNA add T four DNA ligase only after the half linkers and chip DNA fragments a well mixed to prevent concatenation of half linkers Seal the tube with parfum Incubate overnight at 16 degrees Celsius with rotation. Intact Chromatin complexes are then alluded off the bead through the addition of SDS at buffer EB to eluted DNA to lower the SDS concentration at Triton X 100 to quench all the SDS circularization of chromatin complexes is facilitated by the ligation of two half linkers, creating a complete linker sequence at the interaction junction. Ligate elute chromatin complexes at extremely dilute conditions to minimize ligations between different DNA protein complexes at T four DNA ligase only after the eluted chromatic complexes have been well diluted in the reaction mixture After Reverse cross-linking To remove DNA associated protein purifies circularized DNA fragments using phenyl chloroform Extraction, Use KaiGen phase lock gel system for easy separation.
After phenyl extraction, Use glyco blue A DNA carrier to increase mass and visibility of the palate. Half linker oligo nucleotides contain flanking MM E one recognition sites. MM E one A Type two S restriction enzyme cuts 18 and 20 base pairs downstream of its target binding sites to generate short tags of the chromatin fragment producing PET tag linker tag constructs abbreviated as pets.
In addition, each half linker is modified with biotin to enable purification of pet constructs by streptavidin coated magnetic bead. Perform MME one digestion to release tag Linker constructs. Purify the pets by selective binding to strapped Aden beads ligate the pet constructs with adapters for high throughput sequencing.
Use fusion DNA polymerase to amplify the CHI PET Library Fusion. DNA polymerase is used to generate templates with high accuracy and efficiency. Set up PCR reaction cold and add fusion DNA polymerase last.
As the enzyme exhibits three prime to five prime exonuclease activity that can degrade primers in the absence of DN Tps. If the chair pet library is successfully constructed, there should be a prominent, well-defined band of approximately 223 base pairs. Avoid non-specific bands when excising the 223 base pair band from the gel and purify the excised band using the Gel Crush Protocol.
Perform a Quality check using Agilent 2, 100 bioanalyzer with a DNA 1000 kit before high throughput sequencing. We have just demonstrated the CHI PET procedure for unbiased detection of genome-wide chromatin interactions. The quality of CHI PET data depends on the effectiveness of the chip antibody used and sequencing depth.
Here we use 18 to 20 million reads for interaction mapping. This specific protocol has been used to successfully characterize chromatin interaction networks bound by the estrogen receptor and RNA polymerase in the human genome. We foresee this assay to be a powerful tool for future studies after the three dimensional architecture of transcription biology in whole genome context.
We hope this video will be useful to you. Thank you for watching.I.
