The overall goal of the experiment was to identify protein-affiliated DNA fragments throughout the genome, which reflected the status of the chromatin. To achieve this goal, three special operations were performed. First of all, the intact nuclei from plant cells were isolated.
Secondly, modifications to the chromatin were recognized by a specific antibody in situ. Thirdly, the antibody tethering a protein A-transposase fusion protein, Tn5 transposase, cleave the chromatin in situ under the activation of magnesium ions. The DNA fragments and chromatin modification binding sites were then integrated with adapters and made ready for DNA preparation.
Polymerase chain reaction enrichment enhanced generation sequencing. Epigenomics regulation on the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. CUT&Tag technology developed by Henikoff Lab is an enzyme tethering method for chromatin epigenomics profiling at a high efficiency.
Here we employ the cotton leaves as experimental materials, and to perform the chromatin profiling using antibody of Histone H3 Lysine-4 Trimethylation as an example. We provide a refined protocol with video for CUT&Tag performance in plants. For each sample preparation using CUT&Tag, gram of cotton leaf tissue was collected.
The leaf was grind into a fine powder with liquid nitrogen, and transferred into a 50 mL falcon tube containing 25 mL of chilled nuclear isolation buffer A.The buffer solution was mixed gently, and the tube was incubated on ice for five minutes with gentle shaking to disperse the material evenly. It was then spun at 500 relative centrifugal force or rcf, at 4 degrees Celsius, for five minutes, to form the cell pellet. The supernatant was decanted and the pellet was resuspended with 20 mL of chilled nuclear isolation buffer B, supplemented with 0.5%Triton.
The tube was inverted gently to resuspend the pellet completely. The resulting cell lysate was then filtered with a combination of two sieves, a 500-mesh sieve was used on top to remove the larger tissue debris. And a 1000-mesh sieve was used on the bottom to collect the nuclei.
The choice of the appropriate mesh size of the sieves depends on the size of the nuclei of the plants. Sterile filter paper was used on the back side of the sieve to absorb part of the lysate and small cell debris. The lysate containing the nuclei was transferred to a fresh 1.5 milliliters centrifuge tube, and spun at 300 rcf for four minutes, at 4 degrees Celsius degrees Celsius to collect the nuclei.
After centrifugation, a pipette tip was used to remove as much of the supernatant as possible. An amount of 800 microliters, a nuclear wash buffer was added. And the tube was inverted gently to resuspend the nuclei.
The tube was spun again at 300 rcf, for four minutes at 4 degrees Celsius. After a total of three washes, the resulting nuclei were combined in a fresh 1.5 milliliters centrifuge tube, and spun at 300 rcf, for four minutes at 4 degrees Celsius. A pipette tip was used to remove as much the remaining buffer as possible.
The next step was the incubation of the antibody. An aliquot of 50 microliters of nuclei was resuspended in 1 milliliter of antibody buffer supplemented with EDTA, BSA, protease inhibitor cocktail in digitonin. An aliquot of 150 microliters of the nuclei suspension was placed in a fresh 1.5 milliliter tube for one reaction, two microliters of antibody were added to each tube.
In this assay, two biological replicates for the anti-histone-H3-Lysine-4-Trimethylation antibody and immunoglobulin G negative control groups were set up after antibody was added. The solution was mixed gently, and the tubes were left on a horizontal shaker for hybridization overnight at four degrees Celsius in 12 rpm. The next morning, the tubes were taken out and the nuclei were washed using 800 microliters of immuno precipitation wash buffer.
The tube was inverted gently for mixing and it was left on horizontal shaker for five minutes at room temperature in 12 rpm. After washing, the tube was spun at 300 rcf for four minutes at four degrees Celsius. After centrifugation, the supernatant was removed using a pipette tip.
This was repeated for a total of three washes. After the third wash, the tube was spun a 300 rcf for two minutes at four degree Celsius, and a pipette tip was used to remove the remaining buffer as much as possible. To prepare the Tn5 transposase mix for incubation.
One microliter of transposase was added to each 150 microliters of the transposase incubation buffer. We recommend preparing the transposase solution mix first according to the number of reactions. And then adding an aliquot of 150 microliters to each tube.
The solution was mixed gently and left on a horizontal shaker at room temperature in 12 rpm for two hours. The tubes were mixed gently every 10 to 15 minutes. After incubation, the nuclei were washed three times using 800 microliters of immuno precipitation wash buffer at room temperature to remove the unbound Tn5 transposase.
After the third wash, the tube was spun at 300 rcf for two minutes at four degree Celsius, and a pipette tip was used to removed the remaining buffer. For the tagmentation step, 300 microliters of tagmentation buffer was added to the reaction tube. The solution was mixed well and incubated at 37 degrees Celsius for one hour.
After incubation 10 microliters of 0.5 mole per liter EDTA at 30 microliters of 10%SDS were added to determine the reaction. Next 300 microliters of DNA extraction buffer was added. Irregular DNA extraction was performed.
The DNA fragments were dissolved in 25 microliters of sterile water. 24 microliters were used for PCR amplification in NGS library construction. This figure shows the intact nuclei stain with DAPI in imaged, under a microscope obtaining an adequate amount of intact and relatively clean nuclei is the critical step for CUT&Tag.
After PCR, two microliters were transferred and the size of the DNA was checked on 1.5%agarose gel. This figure shows that compare with the immunoglobulin gene negative control. The bulk of the DNA fragments from the Histone H3 Lysine 4 Trimethylation sample should range from 280 to 500 base pairs.
The DNA fragments can be purified and then profiled by high throughput sequencing. This figure shows the results generation sequencing for the anti-histone H3 Lysine 4 Trimethylation antibody compare with the immunoglobulin G negative control groups. This technology generates the DNA libraries at high resolution and exceptionally low background using a small number of cells with a simplified procedure in compared with ChIP-seq.
CUT&Tag is a new method and still in progress.
