Hi.C is a method of identifying long range chromatin interactions in an unbiased genome wide fashion. First cells are fixed with formaldehyde cells are lysed and the DNA is subsequently fragmented with a restriction enzyme. Next, a biotinylated residue is incorporated as the five prime overhangs are filled in blunt and ligation is performed under dilute conditions that favor ligation events between cross-links, DNA fragments.
The library is sheared and the junctions are pulled down with strep havein beads. The purified junctions can subsequently be analyzed using a high throughput sequencer resulting in a catalog of interacting fragments. These results provide us with insights into the folding of chromatin, revealing the presence of chromosome territories, the com compartmentalization of the genome in open and close ch chromatin and evidence for the Fractal Glo organization at the Megabase scale.
Hi, I'm Nka Van Bergham from the Laboratory Field Decker in the program of gene function and expression at the University of Massachusetts Medical School. Hi, I'm Ez Lieberman Aiden of the laboratory of Eric Lander at the Broad Institute of Harvard and the Massachusetts Institute of Technology. I'm Louis Williams from the Molecular biology r and d group at the Broad Institute And then Maxima Mackay from the laboratory of Len Muni from the Massachusetts Institute of Technology.
Today we will show you a procedure for genome-wide chromosome confirmation capture called high C.We use this procedure in our laboratory to experimentally explore the architecture of the human genome. The first part of the procedure we'll show you will take place at UMass Medical School And the second part will be done at the Prude Institute. We'll also show you how we've used the results of the high C experiment to simulate the dynamics of the genome at MIT.
So let's get started. So Let's, let's get started. Hi C begins with cross-linking of cells, which is a common method in chromosome confirmation capture.
To begin grow between two times 10 to the seven and 2.5 times 10 to the seven mammalian cells, either a Durant or in suspension and cross-link the cells lies the cells in 550 microliters of lysis buffer using homogenizer. Spin the chromatin at 5, 000 RPM and wash the pellet twice with 500 microliters of NEB buffer two qu into five numbered tubes and add any B buffer. Two to a final volume of 362 microliters.
Add 38 microliters of 1%SDS mix carefully and incubate a 65 degrees Celsius for 10 minutes. Place tubes back on ice immediately and quench the SDS by adding 44 microliters of Triton X 100 mixed carefully at 400 units of Hindi three and incubate at 37 degrees Celsius overnight while rotating. The next steps are high C specific and include marking the DNA ends with biotin and performing blunt end ligation of cross-linked fragments.
This step will allow ligation junctions to be purified later tube. One should not undergo the biotin elation step and should instead serve as a three C control to ensure that digestion and ligation conditions were optimal to fill in the restriction fragment overhangs and mark The DNA ends with biotin in the remaining four tubes. Add D-A-T-P-D-G-T-P and DTTP to tubes two to five.
Also add biotin elated DCTP and Klino. Mix carefully and incubate for 45 minutes at 37 degrees Celsius. Following incubation, place the tubes on ice, then add 86 microliters of 10%SDS to all tubes to inactivate the enzymes.
Incubate the tubes at 65 degrees Celsius for exactly 30 minutes and place them on ice immediately afterwards. To favor ligation events between cross-linked fragments, the ligation is performed under extremely dilute conditions. Working on ice add 7.61 milliliters of ligation mix to each of five 15 milliliter tubes.
Then transfer each digested chromatin mixture to the corresponding tube. Add 10 microliters of T four DNA ligase to tube one to perform a regular three C ligation. Then add 50 microliters of T four DNA ligase to tubes two to five to perform blunt end high C ligation.
Mix the tubes by inverting them and incubate for four hours at 16 degrees Celsius. Following incubation degrade protein and reverse CNA cross-linking by adding 50 microliters of proteinase K per tube and incubating the tubes overnight at 65 degrees Celsius. Add an additional 50 microliters of proteinase K per tube the next day and continue to incubate a 65 degrees Celsius for another two hours.
Call the reaction mixtures and transfer them to five 50 milliliter to conical tubes. Purify the DNA in these tubes by performing a phenol extraction. Add 10 milliliters of phenol and vortex for two minutes.
Spin the tubes for 10 minutes at 3, 500 RPM and carefully transfer as much of the aqueous phase as possible to a new 50 milliliter tube. Repeat the extraction using phenol chloroform and precipitate the DNA using ethanol after centrifugation of the ethanol precipitated DNA each DNA pellet is dissolved in 450 microliters of one XTE buffer and transferred to a 1.7 milliliter centrifuge tube. Another round of purification is performed by doing two phenol chloroform extractions at 500 microliters of phenol, chloroform and vortex for one minute centrifuge to the tubes for five minutes at 14, 000 RPM and transfer the aqueous phase to a new tube after the second extraction.
The DNA is ethanol precipitated after spinning down the precipitated DNA each DNA pellet is washed once with 70%ethanol and then resuspended in 25 microliters of one XT buffer degrade any RNA that may be present by adding one microliter of RNAs a per tube and incubating the tubes for 30 minutes. At 37 degrees Celsius. Pull the high C contents of tubes two to five, still keeping tube one separate as a three C control.
Now is a good time to examine the quantity and quality of the libraries by running an aliquot in a 0.8%AASE gel marking the ligation efficiency of HI C should be evaluated with a PCR digest assay. The blunt end creation and ligation performed in the HI C procedure will create restriction sites for the endonuclease in HE one amplified fragments from the three C and high C libraries can be digested with Hindi three and NHE one to determine how well the blunt ends were generated and some fragments will not have been ligated. Remove biotin from these unligated ends using the exon nuclease activity of T four DNA polymerase as described in the written protocol.
Purify the DNA again using phenol chloroform extraction and ethanol precipitation and resuspend the DNA pellets in a total volume of 100 microliters of water. If the sample has passed the quality control assays, then the protocol continues with bio timple down and sequencing. Thank you.
To make the BIOTINYLATED DNA suitable for high throughput sequencing, the DNA must be shared to a size of 300 to 500 base pairs with a CAVAS S two instrument. The she DNS are repaired by adding 10 x lation buffer DN TPS T four DNA polymerase T four POLYNUCLEOTIDE kinase clear now DNA polymerase and water incubate the samples for 30 minutes at room temperature following incubation, use a Kaya Gym MinLu column to purify the DNA according to the manufacturer's recommendations. After loading the DNA on the column and washing the column, elute the DNA with 50 microliters of one XTLE buffer twice, then attach DATP to the three promen of the end repaired DNA by adding 10 XNEB buffer, two DATP water and exonuclease deficient cile fragment.
Incubate the reaction for 30 minutes at 37 degrees Celsius to inactivate the cile fragment. Incubate the reactions for 20 minutes at 65 degrees Celsius and subsequently call the reactions on ice using a speed vac, reduce the reaction volumes to 20 microliters. Next, load the DNA in a 1.5%Aris gel with one XTAE and run for 3.5 hours at 80 to 90.
Vols excise DNA fragments between 300 and 500 base pairs and purify them with a Qiagen gel extraction kit using two to four columns depending on the weight of the gel. Combine the yellow eights from the kayak quick columns and bring the final volume up to 300 microliters with one XTLE buffer. Finally, determine the DNA concentration with the QU assay using the qubit and calculate the total amount of DNA in this section of the protocol.
Ligation junctions are purified from the DNA pool, allowing for efficient identification of interacting chromatin fragments by paired end sequencing. Perform all subsequent steps in DNA low bind tubes. Prepare beads for biotin.
Pull down by washing 150 microliters of resuspended magnetic strep A in beads twice with 400 microliters of one x tween buffer. These in future washes consist of five steps, adding buffer to the beads, transferring the mixture to a new tube, rotating the sample for three minutes at room temperature. Reclaiming the beads using a magnetic particle concentrator.
I am removing the supinate reus. Bend the beads in 300 microliters of two x no tween buffer and combine with 300 microliters of high CDNA. Allow the biotin labeled high CDNA to bind to the strep din beads by incubating the mixture at room temperature for 15 minutes with rotation.
Reclaim the DNA bound strp A in beads with a magnetic particle concentrator and remove the supernatant. Wash the beads with 400 microliters of one x no tween buffer, followed by 100 microliters of one x ligation.Buffer. Resuspend the beads in 50 microliters of one x ligation buffer and transfer the mixture to a new tube.
To prepare the DNA for Illumina paired in sequencing, take the total amount of DNA user's input for the biotin pull down, which was calculated earlier using the quant assay and divide it by 20 to estimate the amount of high CDNA that has been pulled down and is available for ligation. Add six P OLS of Illumina paired and adapters per microgram of high CDNA available for ligation. Use 1, 200 units of T four DNA ligase to ligate the adapters to the DNA.
Incubate the two hours at room temperature. Remove non ligated paired end adapters by reclaiming the high CDNA bound beads and washing the beads twice with 400 microliters of one x tween buffer. Wash the beads with 200 microliters of one x, no tween buffer, followed by 200 microliters and then 50 microliters of one XNEB buffer.
Two after the last wash. Resuspend the beads in 50 microliters of one NEB buffer two and transfer to a new tube. To determine the number of cycles necessary to generate enough PCR product for sequencing.
Set up four test PCR reactions with 6, 9, 12, or 15 cycles. Determine the optimal cycle number by running the PCR reactions on a 5 cent poly acrylamide gel and staining with cyber green, ensuring the absence of spurious bands and the presence of a smear between 400 600 base pairs, which is the length of the shared products after ligation to the adapters, amplify the rest of the high C library round stripped, have in beads in a large scale PCR with the optimal number of PCR cycles. Pull the PCR products from the separate wells and reclaim the beads.
Keep 1%of the large scale PCR products separate to run on a gel and purify the remainder of the PCR product with 1.8 times volume and pure beads according to the manufacturer's recommendations. Elute the DNA with 50 microliters of one XTLE buffer and compare 1%of the PU bead purified PCR product to the 1%aliquot of the original PCR product on a 5%poly acrylamide gel. Ensuring the successful removal of the PCR primers sequence, the high C library with Illumina paired end sequencing align each end independently using MAC to identify interacting chromatin fragments.
The following results are expected when the high C protocol is executed technically well and can be considered quality control standards. Quality control steps should reveal that both three C and high C libraries run as rather tight bands larger than 10 kilo bases. A DNA smear indicates poor ligation efficiency.
Typically, ligation efficiency is slightly lower in a high C library as compared to a three C template. High C marking and ligation efficiency can be estimated by digestion of a three CPCR product. Three C junctions are cut by Hindi three and not by NHE one.
The reverse is true for high C junctions. This PCR digest assay shows that 70%of high C amplicons are cut by NHE one and not by HI Hindi.Three. Confirming efficient marking of ligation junctions analysis of the sequenced reads should show that reads from both intra chromosomal and inter chromosomal interactions indicated by the blue and red lines align significantly closer to Hindi three restriction sites as compared to randomly generated reads shown in green.
In a successful experiment, 55%of the alignable read pairs represent inter chromosomal interactions. 15%represent intra chromosomal interactions between fragments less than 20 kilobases apart, and 30%are intra chromosomal read pairs that are more than 20 kilobases apart. This distribution may be sampled prior to high throughput sequencing as a form of quality control.
Cloning and Sanger sequencing around 100 clones is usually sufficient. The chromatin interactions can be visualized as a heat map where the x and Y axis represent low sign genomic order and each pixel represents the number of observed interactions between them. Typically, DNA fragments that are very close to each other in the linear genome will have the tendency to interact frequently with each other.
This is seen in the intra chromosome or heat maps as a prominent diagonal. The following results show different ways of analyzing the data to reveal various levels of genome. Organization plotting the contact probability versus genomic distance shows that the probability of contact decreases as a function of genomic distance eventually reaching a plateau at every distance.
Intra chromosomal interactions shown in the solid line are enriched relative to inter interch chromosomal interactions represented by the dash lines. This directly implies the presence of chromosome territories calculating the observed versus expected number of inter chromosomal context between all pairs of chromosomes reveals preferential association between particular chromosome pairs. Small gene rich chromosomes preferentially interact with each other indicated by the bright red color.
Individual chromosomes can also be examined. The raw heat map can be adjusted using an expected heat map to account for the genomic distance between pairs of loci resulting in an observed over expected heat map. Then a correlation matrix can be produced by correlating the rows and columns of the observed over expected heat maps.
Using correlation analysis, it has demonstrated that the human genome segregates into two compartments. This is illustrated by the plaid pattern. In the correlation heat maps using high C data, new insights were gained into chromatin folding at the megabase scale.
An ordinary polymer model of chromatin packing would suggest that chromatin pack into an equilibrium GLO Ploting contact probability is a function of distance illustrates that contact probability scales as a power law with genomic distance whose slope is approximately minus one. This is not consistent with the behavior of an equilibrium Glo, but does match expectations for an alternative structure known as a fractal glo. Here two gular structures are shown.
Coloration corresponds to distance from one endpoint ranging from blue to cyan green, yellow, orange, and red. And like equilibrium globules, fractal globules lack entanglements in a fractal Glo. Loci to nearby along the contour tend to be nearby in 3D leading to the presence of monochromatic blocks.
Such blocks are not found in the equilibrium Glo. The lack of entanglements can also be demonstrated by removing the force constraining the globules and allowing the globules to expand the fractal glo rapidly decon condenses. Whereas the equilibrium Glo does not because it is too knotted.
We've just shown you how to make an analyzer high C library. When doing this procedure, it's important to remember to do the proper controls to ensure efficient high C ligation and purification. The number of reads will ultimately determine the resolution of the interaction maps.
We have used about 30 million reads to generate interaction maps of the human genome at a resolution of about one megabase. In order to increase the resolution by a factor of N.The number of reads needs to be increased by a factor of n squared. I hope you enjoy learn about the structure of the human genome.
So that's it. Thanks for watching and good luck with your experiments.
