The overall goal of this experiment is to generate next generation sequencing libraries from both the DNA and RNA of small populations of cells without splitting the sample at any point. This method provides researchers with a new approach to study the structural variations of DNA on the expression of genes. When we first thought up Gel-seq, there were no protocols for simultaneously sequencing both the DNA and RNA from the same sample.
Fortunately, there are now three published approaches that solve this problem. The main advantage of this technique is that both genomic and transcriptomic information can be obtained from the same set of cells for negligible added cost. Gel-seq was originally developed with upright cassettes, however, this protocol can be adapted to work with any standard gel electrophoresis cassette.
Prepare the filler, the high density, and the low density precursor mixtures in three tubes. Add the reagents in the order described, and vortex the mixture. Do not prematurely add either APS or Temed.
Wait until directed. Next, degas each gel monomer solution by first inserting a needle through the cap. Then connecting the needle to a house vacuum line, and submerging this assembly in an ultra sonic bath.
Wait about one minute until no more bubbles emerge from the liquid. Next, to the filler precursor mixture, add the Temed and the APS and vortex briefly. Within three minutes, use a one millimeter pipette to add six millimeters to each gel cassette without spilling.
Ensure the fluid is level by positioning the cassettes upright on a level table. Then, gradually top off the cassettes with degassed, ionized water, being careful to minimize the mixing of the polymerizing mixture with the water. Then, allow the polymer to cure for an hour to overnight.
Once the polymer is cured, dump out the water overlay, and from six inches away use a gentle stream of compressed air to dry the gel's surface. Next, add Temed and APS to the high density gel precursor, and vortex briefly. Within three minutes, transfer 320 microliters to each cassette, and spread it over the filler gel layer by rocking the cassette back and forth a few times.
Then, top off the cassettes with deionized, degassed water, and allow the polymer to cure for about an hour. Once cured, dry off the polymer as before. Finally, add Temed and APS to the low density gel precursor mixture, and vortex it.
Then, quickly fill the remainder of the cassettes with this mix, and insert the gel combs before it starts to cure. Now, add the excess of reserve precursor over the comb to be adsorbed during polymerization. After curing, the cassette is ready for use.
After converting the RNA to cDNA, use a gel to recover the fraction of interest. First, thoroughly clean the electrophoresis chamber using several milliliters of a DNA removal product, and a lint-free wipe. Then, fill the chamber with clean, zero point five XTBE, and place the entire apparatus in a 254 nanometer, UV-crosslinking oven, and sterilize for 15 minutes.
Now, insert a Gel-seq cassette into the electrophoresis chamber and lock it in place. Then, slowly remove the gel comb by gently pulling upwards without tearing the gel. Now, load samples using a pipette.
Fully insert the tip into the wells when loading samples to minimize the chance of contamination between wells. Apply an electric field of 250 volts across the Gel-seq cassette for 30 minutes to separate the gDNA from the cDNA/RNA hybrids. After loading, running, and collecting the gel use a scalpel to cut the gel in half just below the high density layer, and discard the filler gel half.
Then, gently peel the remaining gel off of the cassette making careful use of a scraping tool. After verifying separation using a gel doc, use a transilluminator to excise the bands. The gDNA should be located at the start of the low density gel, and the cDNA should be at the interface of the low and high density regions.
Now, use a scalpel to cut out the regions of the gel containing gDNA and cDNA. Also remove the same sections from the negative control lane. Using blunted tweezers, gently transfer the sections of the gel into strip tubes.
If the gel breaks, find and transfer all the broken pieces to the tube. Next, grind down each gel using circular movements, and pressing with a pipette tip. Before removing the tips, add 40 microliters of nuclease-free water to the gDNA, and 80 microliters to the cDNA samples.
Then, carefully remove and discard the tips. Now, secure the strip tubes to a vortex mixer at 37 degrees Celsius and shake them for eight to 12 hours. The DNA will thus elude from the gel.
After the shaking, pipette the samples into an eight micron mesh filter plate. Then, spin the plate at 2, 600 g for five minutes to strain out the gel fragments. Next, lift the mesh filter plate away from the housing plate and use a pipette to transfer the gel-free samples from the plate to new 200 microliter strip tubes.
To each sample containing gDNA, add one microliter of protease, and pipette up and down to mix well. Then, incubate the samples at 50 degrees Celsius for 15 minutes. Follow with a heating activation at 70 degrees Celsius for 15 minutes.
This nucleosome depletion step is critical. Next, use an 18 gauge needle to poke a hole in the cap of every sample tube. Then, use a vacufuge to reduce the gDNA samples to five microliters, and the cDNA samples to 10 microliters.
If the volume drops below the target, add nuclease-free water as needed. gDNA and cDNA/RNA hybrids were physically separated using the Gel-seq device. The negative control has no auto fluorescence of the gel at the interfaces.
The DNA ladders show only a dark band at the interface between the low and high density membranes, revealing that small fragments can pass through the low density gel. A biological sample from 500 PC3 cells shows the desired separation of genomic DNA and cDNA/RNA hybrids. A dark band at the top of the low density membrane is megabase-scale genomic DNA while the cDNA/RNA hybrids are stacked at the interface of the low and high density regions.
The small sub-100 base pair fragments are off target products generated from primer oligonucleotides during reverse transcription and are to be expected. Libraries were generated from 500 PC3 cells or 750 HeLa cells. Fragment distributions from the matched libraries generated from Gel-seq were compared to unmatched samples generated with standard protocols.
The distribution of nucleotide fragments was similar with either method. Sequencing results of the PC3 cell library were compared with traditional methods. Genome-wide copy number variations were compared.
Statistically, the two methods garnered similar results. After watching this video, you should have a good understanding of how to generate paired next generation sequencing libraries for both DNA and RNA from the same starting sample. As with any low input sequencing experiment, it's imperative to remember to maintain a clean working environment.
You should pay careful attention to sterilizing tubes and cleaning gloves. Even with these precautions, experiments occasionally fail due to contamination. An important note, the ubiquity and safety of polyacrylamide gels makes it easy to forget that the fabrication precursors are dangerous neurotoxins.
You should always work with these reagents in a fume hood and immediately clean up any spills. We designed Gel-seq to be simple for researchers to implement in their own labs, and we hope this will facilitate the rapid adoption of this technology.
