This method can help answer key questions in the epigenomics field by demonstrating how to design and implement a sequencing-based tool to assess the impact of environmental factors on DNA methylation. The main advantage of this technique is that it can provide a more comprehensive and quantitative method to assess DNA methylation levels compared to array-based platforms. The implications of this technique extend toward identification of DNA methylation changes due to different types of physiological processes in any organism whose sequence data are available.
Though this method can provide insight into the impact of stress on DNA methylation, it can also be applied to other environmental factors or physiological conditions, such as the exposure to environmental toxicants in high-fat diet and conditions such as diabetes and cardiovascular disease. After shearing DNA and verifying the size via bioanalyzer, add 180 microliters of DNA-binding magnetic beads to each sample, and incubate at room temperature for five minutes. Pellet the beads on a magnetic plate, and remove the supernatant.
Resuspend the pellet in 200 microliters of 70%ethanol. Next, use a magnetic plate to pellet the beads, and remove as much ethanol as possible. Dry the beads in a heat block at 37 degrees Celsius for three to five minutes.
Then, resuspend the pellet in 44 microliters of nuclease-free water, and collect approximately 42 microliters of the supernatant. After ligating the adapters and verifying ligation via bioanalyzer, transfer the samples to low DNA-binding microcentrifuge tubes. Then, use a heated vacuum concentrator to reduce the sample volume below 3.4 microliters.
After this, add 5.6 microliters of Methyl-Seq Block Mix to each sample, and incubate them in a thermal cycler. Prepare Capture Library Hybridization Mix according to the text protocol. Then, add 20 microliters of Capture Library Hybridization Mix to each sample, and incubate at 65 degrees Celsius for 16 hours.
Toward the end of the incubation period, prepare streptavidin magnetic beads by adding 50 microliters of streptavidin magnetic beads per sample to a new eight-well strip tube. Wash the beads with 200 microliters of Methyl-Seq Binding Buffer. Place the strip tube on a magnetic plate, and remove the supernatant from the beads.
Then, resuspend the beads in 200 microliters of Methyl-Seq Binding Buffer. Add the samples to the washed streptavidin beads, and incubate them at room temperature for 30 minutes on a rotating mixer. While the samples mix, aliquot 200 microliters of Methyl-Seq Wash Buffer Two into triplicate wells of a 96-well plate and pre-warm to 65 degrees Celsius.
After the samples incubate, pellet the magnetic beads with a magnetic plate and remove the supernatant. Then, resuspend the beads in 200 microliters of Methyl-Seq Wash Buffer One. Incubate the beads for 15 minutes at room temperature, and use a magnetic plate to remove the supernatant.
After this, resuspend the bead pellet in 200 microliters of pre-warmed Wash Buffer Two. Then, incubate the beads in a thermal cycler before using a magnetic plate to pellet the beads. Add 20 microliters of Methyl-Seq Elution Buffer to the washed beads, and incubate them at room temperature for 20 minutes.
Use a magnetic plate to pellet the beads, and transfer the supernatant to a new strip tube. First, add 130 microliters of bisulfite conversion reagent to the previously collected supernatant. Divide the 150-microliter reactions equally into two wells.
Then, incubate the reactions in a thermal cycler. Use 600 microliters of Binding Buffer to bind the samples to the spin columns, and wash the columns with 100 microliters of Wash Buffer. Then, centrifuge the columns, and discard the flow-through.
After this, add 200 microliters of Desulphonation Buffer to the columns. Incubate the columns for 15 to 20 minutes at room temperature. Wash the columns twice with 200 microliters of Wash Buffer.
Then, add 10 microliters of Elution Buffer to the column. Incubate at room temperature, and centrifuge. Prepare the PCR reaction master mix according to the text protocol.
Then, add 82 microliters of the master mix to each sample, and incubate the samples in a thermal cycler. First, prepare the PCR Master Mix Two on ice according to the text protocol. Then, to each sample, add 25.5 microliters of PCR Master Mix Two and five microliters of commercial indexing primers, and incubate the samples in a thermal cycler.
Assess the DNA concentration of the samples with high-sensitivity DNA detection reagents on a bioanalyzer. The Methyl-Seq libraries are then submitted for sequencing on the next-generation sequencer. After bisulfite converting and PCR amplifying validation samples, prepare a master mix with Binding Buffer, water, and streptavidin-coated sepharose beads.
Then, add 75 microliters of the master mix and five microliters of the nested PCR product to a 96-well plate, and shake the plate on a plate shaker for 15 to 60 minutes. While the plate shakes, add 12 microliters of primer to the wells of a pyrosequencing assay plate. After shaking, place a vacuum tool in a trough filled with water, then place the tool in the plate with binding reactions.
Next, submerge the vacuum tool in half-filled troughs containing 70%ethanol, sodium hydroxide, and Tris-acetate buffer. Disconnect the vacuum, and place the vacuum tool in the HS pyrosequencing assay plate to transfer the beads. Place the plate on a heat block, and incubate at 80 degrees Celsius for two minutes.
Finally, allow the plate to cool for five minutes before beginning the pyro program. In this protocol, analysis of the Methyl-Seq libraries provided a ranked list of differentially methylated regions, or DMRs, between stressed and unstressed animals and a graphical plot of the DMRs. Validation via bisulfite pyrosequencing showed that the stressed animals all exhibited higher methylation levels across all CpGs than the unstressed animals.
Methylation levels at CpG 10 were also compared to the mean three-week CORT levels for each animal. The results indicate that there is a modest correlation between the endocrine and methylation data. While attempting this procedure, it's important to remember to compare the increase in the average DNA size between DNA shearing and adapter ligation steps and to ensure sufficient library quantity prior to sequencing.
Following this procedure, other similar approaches can be implemented in order to answer novel questions, such as the impact of environmental toxicants on rat neurodevelopment. After its development, this technique provided the means for researchers in the field of epigenetics to explore genome-wide DNA methylation changes in any non-model or model organisms where genomic sequences are available. Don't forget that working with the temperature-sensitive enzymes requires storage on ice while in use and storage in the minus 20-degree freezer.
The RNA beads used for target capture require thawing and storage on ice while in use and long-term storage in the minus 80-degree freezer.
