The overall goal of this procedure is to PCR amplify retroviral integration sites from bulk genomic DNA, and sequence the resulting DNA library, such that precise integration locations can be mapped to a reference genome. This method can help answer key questions in basic and clinical retrovirology by analyzing the distribution of integration sites throughout a host genome. The main advantage of this technique is the DNA sequencing depth, and the fact that many samples may be multiplexed in one sequencing run.
One day prior to transfection, plate 3.3 times 10 to the sixth HEK293T cells in 10 milliliters of supplemented Dulbecco's Modified Eagle's Medium in each of five 100 millimeter dishes. The next day, use commercially available transfection reagents or calcium phosphate to transfect the cells with 10 micrograms of plasmid carrying full-length retroviral molecular clones or nine micrograms of enveloped elided single round vectors plus one microgram of a VSV-G expression construct. Add the mixture to the cell media dropwise.
Following transfection, incubate the cells at 37 degrees Celsius in a humidified cell culture incubator with 5%carbon dioxide. After approximately 48 hours, harvest the virus-containing cell media using a volumetric pipette, and pass it through a 0.45 micron filter by gravity flow. Next, concentrate the virus by ultracentrifugation at 200, 000 Gs for one hour at four degrees Celsius.
Following centrifugation, remove the supernatant until approximately 500 microliters of DMEM FPS remains, and resuspend the virus pellet. Add 20 units of DNase and incubate for one hour at 37 degrees Celsius. Determine the p24 concentration using an HIV-1 p24 antigen capture kit according to the manufacturer's instructions.
The day before infection, plate 3.0 times 10 to the fifth HEK293T cells in 2.5 milliliters of DMEM FPS per well in a six well plate and incubate overnight in a tissue culture incubator. Infect the cells with a final viral p24 concentration of 500 nanograms per milliliter in 500 microliters of fresh DMEM FPS. Incubate for two hours in a tissue culture incubator.
After two hours, add two milliliters of DMEM FPS prewarmed to 37 degrees Celsius to each well and return the plate to the incubator. 48 hours post-infection, remove the media and wash the cells with two milliliters of phosphate-buffered saline. Harvest the cells by adding 0.5 milliliters of Trypsin-EDTA prewarmed to 37 degrees Celsius, and after a few seconds, visually inspect the wells for cell dislodgement.
Next add two milliliters of prewarmed DMEM FPS and resuspend the cells by gently pipetting up and down 10 times with a volumetric pipette. Transfer the solution to a 75 square centimeter tissue culture flask containing 18 milliliters of prewarmed DMEM FPS, and return to the tissue culture incubator. A minimum of five days after infection, harvest the cells as before and resuspend in five milliliters of prewarmed DMEM FPS by pipetting.
Centrifuge the solution for five minutes at room temperature at 2, 500 Gs, then aspirate and discard the supernatant. Lastly, extract genomic DNA from the cell pellet using a commercially available kit. Elute the DNA from the supplied ion exchange column with 200 microliters of millimolar Tris-HCL at pH 8.5.
After sonication of the genomic DNA, purify the sonicated DNA using a PCR purification kit. Next, repair the DNA using an end repair kit. After purification of the DNA as before, A tail the DNA using Klenow Exo-Minus enzyme.
Alternatively, for fragmentation by restriction endonuclease digestion, cut genomic DNA with a cocktail of enzymes that generate 5'TA overhangs, as well as an incompatible enzyme such as BglII that cleaves downstream from the upstream viral LTR. Cut 10 micrograms of genomic DNA in a final volume of 100 microliters. Incubate overnight at 37 degrees Celsius.
Ensure that the final DNA is cleaned up using the PCR purification kit. The linker to be used with sonicated DNA must contain a compatible T 3'overhang, while the linker for MseI digested DNA must contain a compatible 5'TA overhang. Begin by annealing 10 micromolar of the short and long linker strands in 35 microliters of Tris-HCL EDTA buffer by heating to 90 degrees Celsius and slowly cooling to room temperature in steps of one degree Celsius per minute.
Next, prepare at least four parallel ligation reactions per genomic DNA sample containing 1.5 micromolar ligated linker, one microgram of fragmented DNA, and 800 units of T4 DNA ligase in a final volume of 50 microliters. Ligate overnight at 12 degrees Celsius. For samples prepared by sonication, digest the purified ligation reaction with a restriction enzyme that cleaves downstream from the upstream LTR.
Use 100 units of the appropriate enzyme under the manufacturers recommended conditions overnight. Ensure that final products are purified using a PCR purification kit. Prepare the first round PCRs using the reagents shown here.
Run the first round PCR using the following parameters:94 degrees Celsius for two minutes, followed by 30 cycles of 94 degrees Celsius for 15 seconds, 55 degrees Celsius for 30 seconds, 68 degrees Celsius for 45 seconds. Finish with 68 degrees Celsius for 10 minutes. Pool the first round reactions and purify the DNA as before.
Then prepare the second round PCRs containing the reagents shown here. Use the same cycling parameters as for the first round PCR. After pooling and purifying the DNA from the second round reaction, perform quality control and next generation sequencing, and analyze integration sites using the detailed instructions in the written portion of the protocol.
Integration sites from libraries prepared by digestion with restriction enzymes, as shown on the left, or sonication, as shown on the right, were aligned using web logo software. Each dilution in the titration series is depicted, from neat DNA at the top of the figure, to the maximum dilution of one to 15, 625 at the bottom. The degree of certainty decreases with increasing dilution of infected cell DNA.
This image shows the sequence logo for the matched random control of 50, 000 unique genomic sites. Note that the random sites aligned from the MRC dataset, by contrast, failed to generate appreciable levels of base preferences. Once mastered, integration site libraries can be prepared from genomic DNA in three days.
Integration site mapping, from initial infection to completed bioinformatics, can therefore be completed in about two weeks. Following this procedure, thousands or even millions of unique retroviral integration sites can be cataloged, and diverse analyses can be conducted to quantify associations among insertion sites and genomic annotations.
