Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

We are conducting research in functional genomics with a focus on identifying and characterizing the functional elements within the human genome. Our main interest is in exploring the part of the genome that haven't been well-allocated yet to better understand their potential roles and how they might contribute to human biology and disease. Current reverse genetics tools have some limitations, such as non-specific effects and high cost in creating complex libraries of SSIs or SGIs.

Another challenge is that this method may only target allocated genomic elements, leaving a large portion of the genome underexplored. The protocol developed here offers a new approach for identifying previously unknown genomic elements, including novel functional exons in both protein coding and long coding eyes. To begin, use genomic DNA isolated from a lentivirus-based insertional mutagenesis cell library as a template for a linear PCR in a 50 microliter reaction.Then.

add the other required reagents shown here in the PCR tube. Perform 50 cycles of linear PCR in a standard thermocycler under the given PCR conditions. Transfer the PCR products to a new clean 1.5 milliliter centrifuge tube.

Mix the PCR products thoroughly with 10 microliters of biotin-streptavidin magnetic beads and incubate the mixture for two hours at room temperature, shaking at 400 RPM in a metal bath. Then, use a magnetic stand to collect the beads and carefully remove the supernatant. Wash the beads with 300 microliters of binding or wash buffer six times, discarding the supernatant after each wash.

For the second strand synthesis, resuspend the beads in a 24 microliter reaction volume containing Klenow fragment buffer, DNTP mixture, and P5N6 primer in a PCR tube. Pre-incubate the mixture at 15 degrees Celsius for 20 minutes in a PCR thermocycler, then add two units of Klenow fragment to the reaction mixture. Incubate the mixtures in a PCR thermocycler under the given conditions.

Then, transfer the PCR products into a new clean 1.5 milliliter centrifuge tube. Use a magnetic stand to capture the beads and discard the supernatant. After that, wash the beads gently with 300 microliters of DNase and RNase-free distilled water at four degrees Celsius.

Collect the beads and discard the supernatant. Resuspend the beads in a 25 microliter PCR reaction volume containing Taq buffer, Taq DNA polymerase, primer P5, DNTP mixture, and primer 3-LTR_Nest in a PCR tube. Perform the PCR in a thermocycler with the settings shown here.

Use five microliters of the PCR products as the template for the next round of PCR reaction with primer Illumina P5 and primer Illumina P73-LTR. Perform the PCR in a thermocycler under the given conditions. Then, transfer the PCR products to a clean 1.5 milliliter centrifuge tube.

After equilibrating the beads from two to eight degrees Celsius to room temperature for 30 minutes, mix the beads thoroughly by inverting or vortexing. Pipette 1.2 times the volume of the beads into a clean 1.5 milliliter centrifuge tube. Add the PCR products to the beads and mix thoroughly by pipetting up and down 10 times.

Incubate the beads to allow DNA to bind to them. Then, place the samples on a magnetic stand and remove the supernatant once the solution is clear. Now, wash the beads with 200 microliters of 80%ethanol.

Incubate at room temperature for 30 seconds and remove the supernatant. Air dry the beads at room temperature with the lid open for approximately five minutes. Now, remove the tube from the magnetic stand.

Add 22 microliters of DNase and RNase-free distilled water. Mix thoroughly by pipetting up and down 10 times and let it sit at room temperature for two minutes. Place the tube back on the magnetic stand until the solution is clear.

Then, transfer 21 microliters of the supernatant to a clean 1.5 milliliter centrifuge tube. To measure concentration with the fluorometer, prepare the working solution by mixing the reagent and buffer in a one to 200 ratio. Mix 10 microliters of standard one and 10 microliters of standard two, each with 190 microliters of working solution, to prepare the assay standards.

Gently vortex the mixtures for two to three seconds. Next, prepare the assay sample by mixing one microliter of the purified PCR products with 199 microliters of working solution. Gently vortex the mixture for two to three seconds.

Incubate the assay standards and assay samples for two minutes at room temperature, keeping them protected from light, then, turn on the fluorimeter. Select the double-stranded DNA high sensitivity assay program from the main menu. For calibration, insert standard one into the machine and press the read standard button to measure the first standard.

Then, insert standard two and press the read standard button to measure the second standard. For sample measurement, press the run samples button to go to the sample measurement page. Insert each tube containing the assay sample and press the read tube button to obtain the concentration.

In the low-quality sample, a dominant peak around 83 base pairs indicates adapter dimer contamination. In contrast, the high-quality NGS library displays main peaks between 150 and 1, 500 base pairs, reflecting the profile of a successful library.