The overall goal of this procedure is to label and capture five hydroxyethyl cytokine, a newly discovered DNA modification first fragment the genomic DNA by sonication, then perform a beta gluc glucose transferase reaction to transfer azide glucose moieties to five hydroxyethyl cytidine residues on the DNA using Qlik chemistry. Attach a salide biotin linker to the Azid group to produce biotinylated DNA. Next selectively capture the five hydroxyethyl cytidine containing DNA fragments on streptavidin beads in a density independent manner.
The purified five hydroxyethyl cytidine enriched DNA fragments can be used for downstream analyses including next generation sequencing. The main advantage of this technique over the existing approach like antibody antibody based approach is that the capture of phy hydroxyethyl cytosine is independent of PHY C density. Eugene Lee, a senior research associate in the lab, will demonstrate this protocol.
First, sonicate the genomic DNA to a size range suitable for the genome-wide sequencing platform. Verify the size distribution of the fragmented genomic DNA on a 1%aose gel. Now determine the starting DNA amounts based on the abundance of five hydroxyethyl citabine in genomic DNA.
Since the five hydroxyethyl citabine vary significantly among different tissue taps, starting DN amount will depend on the level of five C of the samples. Please refer table one in the manuscript for examples. The first step of the labeling process is to, it's a basical transfer reaction to transfer AZA glucose Es to the five C residues on the fragmented genomic DNA.
Combine the reaction components and mix by pipetting, then incubate the enzyme reaction in a 37 degree Celsius water bath. For one hour. Pass the reaction through a kayak quick nucleotide removal kit using 10 micrograms of DNA per column elute, the DNA with 30 microliters of water per column and pull the samples In the second step of labeling process.
I salify the bing linker is attached to the eza group by click chemistry. For the biotin elation reaction, add five microliters of biotin conjugate working solution per 30 microliters of DNA solution. Incubate in a 37 degree Celsius water bath for two hours.
Process the reaction through a kayak, quick nucleotide removal kit and dilute the DNA in 100 microliters. Then quantify the recovered DNA using NanoDrop. Start with 50 microliters of strep adin dyna beads.
Perform three washes as per manufacturer's instructions, and collect the beads on a magnetic stand. Now dilute the biotinylated DNA with 100 microliters of two times BNW buffer and add the sample to the washed beads. Incubate for 15 minutes at room temperature with gentle rotation.
Harvest the beads with a magnetic stand and wash three times next to elute, the DNA. Add 100 microliters of freshly prepared 50 millimolar DTT and incubate at room temperature under gentle rotation for two hours. Separate the beads using a magnetic stand.
Then collect the EENT containing the target DNA, and load the sample onto a microbio spin six column. To remove the DTT further purify the sample on a cogen column and elute DNA in 10 microliters of EB buffer, quantify DNA using a qubit, fluorimeter, or NanoDrop. If the concentration is higher than 20 nanograms per microliter, this 1%arose gel shows sonicated fragments of DNA, isolated from human IPS cells.
For downstream applications like next generation sequencing, the typical fragmentation size is around 300 base pairs. Note that when the quality of genomic DNA is high recovery yields after the beta GT and biotin reactions are around 60 to 70%However, the pull down yields vary significantly with different tissue types, depending on the five hydroxyethyl cytokine levels of the samples. Typically, the capture efficiency for brain genomic DNA is around four to 9%and in some extreme cases, the efficiency may reach up to 12%For ES cells, the average capture efficiency is around two to 4%in contrast to around 0.5%For neuron stem cells, the lowest deficiency seen so far was for genomic DNA from cancer cells.
Following these procedures, other methods like next generation sequencing technique could be performed to answer additional questions like the genome-wide distribution of five GC modifications.
