The overall goal of this protocol is to detect genomic DNA sequences associated with proteins of interest in animal cells by high throughput sequencing. This technique can help answer key questions in the study of nuclear organization and transcription or regulation in mammalian cells, such as identifying genomic regions associated with the nuclear amyloid or with specific transcription factors. The main advantages of this technique are that high throughput sequencing provides a higher resolution than the original DamID asset, using DNA microarrays, and that this technique enables cross-analysis with our high throughput sequencing data.
Demonstrating the procedure will be Brennan Olson, a technician from our laboratory. To begin, follow the text protocol to clone the gene of interest into the DamID vector. Next, generate lentiviral stocks expressing DamV5 POY and V5-Dam using a lentiviral expression system.
Cotransfect the DamID vector with the lentiviral packaging plasmids following the forward transfection procedure according to the manufacturer's protocol. Be sure to include a filtration step using a zero point four five micron PVDF filter. Now, prepare to infect the cells with the lentivirus.
One day in advance, pass the adherent cells on six-well plates. Continue using antibiotics. The goal is to have 50%confluence the following day for the infection.
The following day, remove the frozen DamV5 POY, and V5-Dam lentiviral supernatants from the freezer, and place them in a 37 degree Celsius waterbath to thaw. On the culture plates, remove the growth media and replace it with half a milliliter of fresh growth media without antibiotics. Then, load the wells with a milliliter of thawed lentivirus.
Load two wells with V5-Dam, two wells with DamV5 POY, and two wells without lentivirus as a negative control. Swirl the solution on the cells and return the plate to the incubator. 24 hours later, replace the viral suspensions on the cells with two milliliters of growth media without antibiotics.
Then, continue the incubation for 48 more hours. Two days later, complete this process by following the text protocol to isolate the genomic DNA, which should take about an hour. Then, amplify the adenine methylated DNA fragments from the genomic DNA.
This process takes about two to three days, and the details are covered in the text protocol. For this procedure, first fragment the amplified adenine methylated DNA. Before using the DNA Fragmentase, it is important to first measure its strength to determine the appropriate digestion time.
Use any available purified methyl PCR products for this diagnostic test. Start by setting up a master mix composed of six micrograms of DNA, 12 microliters of 10X Fragmentase buffer, and fill it with water to 114 microliters. Then, vortex the Fragmentase stock for three seconds and add six microliters into the master mix for a total of 120 microliters.
Then, vortex the master mix for three seconds. Now, fill five reaction tubes with 20 microliter aliquots of master mix and incubate all six tubes at 37 degrees Celsius. Remove one tube after five minutes, and add five microliters of EDTA to stop the reaction.
Do this every 10 minutes until all the reaction tubes are collected. Now, analyze 12 point five microliters from each tube, which corresponds to about half a microgram of DNA, and the same amount of undigested DNA on an agorose gel. From the gel, determine the minimal time needed to digest the majority of the smear to around zero point two kilobases.
Use this as the maximum digestion time. Now, set up the actual fragmentation, like the pretest, and stop digestions at six evenly spaced out time points, including five minutes, and calculate maximum time. Following the digestion, pull the six digestions and purify them using a PCR purification kit, or solid phase reversible immobilization beads.
Dilute the purified DNA in about 15 microliters of EB buffer. The next steps are to repair the ends of the fragmented DNA, add adenine overhangs, ligade on the sequencing adapters, and convert the Y-shaped adapters to double stranded DNA. These steps are all detailed in the text protocol, and shouldn't take more than two days to complete.
After ligating sequencing adapters to fragmented DNA, and converting Y-shaped adapters to double stranded DNA, select fragments of the desired size. Begin with preparing a two percent agorose gel in TAE, and, taking precautions, add ethidium bromide to a final concentration of 500 nanograms per milliliter. Make enough lanes for all the samples and two ladders to be bordered by empty lanes.
Now, add eight microliters of loading dye to 10 microliters of each DNA eluate and prepare the 1 Kb Plus ladder with 6X loading dye and water in a ratio of one to one to four. Then, load the gel lanes in the following order:DNA ladder, DNA eluates, and another DNA ladder. Once loaded, run the gel at 120 volts per 60 minutes.
Later, put on safety glasses and a face shield to briefly view the gel with UV light. Check the DNA ladders to see if the gel has run out far enough so that three gel slices can be excised between 300 and 400 base pairs. Next, cut out the bands between 300 and 400 base pairs from each lane, always using a new blade for each lane.
Remove as little of the gel as possible to make each gel slice ideally under 100 microliters, and transfer each to its own microfuge tube. Next, extract the DNA from the gel slices according to the text protocol. Continue following the text protocol on how to enrich the sequencing adapter modified fragments from the extracted DNA.
Then, get a high quality analysis of the purified DNA by a Bioanalyzer, and if it meets expectations, submit it for sequencing. Lentiviruses were engineered to express a Dam-V5-lamin B1 fusion protein, which was verified to be colocalized with the endogenous lamin B1 protein by amino fluorescence staining. This lentivirus and a control lentivirus expressing Dam-V5 fusion protein were both used to infect mouse C2C12 myoblasts.
DNA was collected and amplified for adenine methylated fragments, as described, and then sequenced. Genome NL interaction maps were then constructed for chromosome one. One track shows the log2 RPKM ratios.
The other track shows the lamina associated domains in black. These are areas that are significantly more associated with Dam lamin B1 than with control Dam. Once mastered, this technique can be done within two weeks, if performed properly.
After watching this video, you should have a good understanding of how to detect DNA sequences associated with proteins of interest by high throughput sequencing, coupled to DamID. Don't forget that working with ethidium bromide and UV lights could be extremely hazardous, and precautions such as waiting for the gel to cool down before adding ethidium bromide, and wearing eye protection and a face shield should always be always taken while performing this procedure.
