Hi, in this video we'll show you how to explore the m6A and m5C epitranscriptomes upon viral infection. To start the experiment, you'll need 50 million cells for the noninfected and 50 million for the infected condition. In order to obtain a high quality dataset upon analysis, you'll need to achieve a universal infection.
Here we infect these cells with an HIV-based vector and measure the virally encoded GFP by FACS. In this case, we had 80%of infected cells and proceeded with the workflow starting with total RNA extraction. So we divided the cells in aliquots of 10 million each and lyse them in 1 mL of Trizol.
We then added 200 microliter of chloroform to the cell lysate and mixed by inversion. At this point, we let the lysate sit for three minutes at room temperature and then centrifuge all samples at high speed for 15 minutes at four degrees. You'll expect to see a phase separation in three different phases, as it's shown here.
RNA is seated in the upper phase, so carefully transfer the aqueous phase into a fresh tube by paying attention in not to transfer the organic layer. At this point, add 0.5 mL of isopropanol to the aqueous phase and mix carefully by inversion. Samples can then be incubated one hour at 80 degrees to ensure RNA precipitation.
Upon centrifugation at high speed, you should be able to see a nice RNA pellet as is shown here. Carefully discard the supernatant either by inversion or by pipetting, paying attention in not disrupting the pellet. Then wash the RNA pellet with one mL of 75%molecular grade ethanol.
And vortex the sample briefly. Upon centrifugation, make sure you retrieve a nice white RNA pellet. Discard the supernatant and let the RNA pellet dry for 15 minutes at room temperature.
Resuspend each pellet in 20 microliter of water and pool all sample from the same condition together. Total RNA is now ready for mRNA isolation by poly-A selection. For each sample of 75 microgram of RNA, prepare 200 microliter of oligo-DT magnetic beads and wash them in one mL of binding buffer.
Place the tube on a magnetic stand and allow the beads to bind to the magnet during one mix. Discard the supernatant and repeat the procedure for a second wash. Then remove of the beads from the magnetic stands and carefully resuspend them in 100 microliter of binding buffer.
At this point, prepare the RNA sample by dividing them in 75 microgram of RNA per aliquot resuspended in 100 microliter of water. Add 100 microliter of binding buffer and incubate for two minutes at 65 degrees. Then spin down the samples and incubate on ice.
Add then 100 microliter of washed magnetic beads to each RNA sample and allow binding on a rotating wheel for 15 minutes at room temperature. Upon incubation, place the tube back into a magnetic rack and let them incubate for one minute. Then recover the supernatant into a fresh tube and keep it aside for a second round of isolation.
Wash the beads twice with 200 microliter of washing buffer and then proceed to elution. In order to elute poly-A selected RNA from the magnetic beads, add 20 microliter of ice-cold TRIS HCl to each sample and pipette carefully. Then incubate your samples at 80 degrees for two minutes to release the RNA from the beads.
Transfer the supernatant containing poly-A selected RNA to a fresh tube, being careful in avoiding beads carryover. The elution step as well as the isolation step should be repeated twice in order to increase mRNA yield. mRNA is now ready either to enter the meRIP-Seq pipeline at the fragmentation step or go directly to bisulfite conversion.
The first step to enter the meRIP-Seq pipeline consists in mRNA fragmentation. For this, you need five microgram of mRNA divided into aliquots of 18 microliter in 0.2 mL tubes. In order to ensure consistent data, it is critical to work quickly and with only few sample at the time.
For this experiment, fragmentation has been optimized in order to obtain fragment of a size between 115 and 200 nucleotides. Then add two microliter of fragmentation reagent to each tube containing 18 microliter of mRNA being careful in depositing the droplet on the tube wall. Spin down all samples in order to allow contact between the reagent and the RNA at the same time and incubate at 70 degrees for 15 minutes.
Once the incubation is over, stop the reaction by adding two microliter of 0.5 M EDTA. At this point, you can purify your fragmented mRNA with your method of choice and proceed to a RNA quality control by fragment analyzer. This step is optional, but it'll allow to assess the size of the fragment before engaging RNA precipitation.
As you can see here in the fourth lane, we obtain for both our sample fragment of around 150 nucleotide each. We then proceeded with meRIP-Seq. The first step consists in couple A/G magnetic beads with either anti-m6A antibodies or IgG controls.
For each reaction plan, transfer 25 microliter of magnetic beads into a fresh tube. Please note that you will need at least one m6A-specific condition and one control condition. Wash each 25 microliter of beads with 250 microliter of IP buffer.
Gently resuspend the beads by pipetting up and down and place the tube on the magnetic rack for one minute in order to allow bead separation. Remove and discard the supernatant, paying attention in not aspirating the magnetic beads and repeat the wash step once. Then resuspend the magnetic beads in 100 microliter of IP buffer per each 25 microliter of original volume of beads.
Prepare and label one tube per each condition. In our case, we will have two tubes for the infected condition, One for m6A and one for IgG control, and two tubes for the noninfected condition. Then add 100 microliter of washed beads in each tube.
Beads are now ready to be coupled with specific antibodies or IgG controls. Add five microgram of either specific anti-m6A antibody or anti-IgG control antibody to each tube containing 100 microliter of washed magnetic beads. Allow antibody-beads conjugation by incubating the tube 30 minutes on a rotating wheel.
Meanwhile, proceed with sample preparation. For each planned condition, either m6A-specific or IgG control, your starting material will be 2.5 microgram of fragmented mRNA resuspended 100 microliter of water. To have a final reaction volume of 500 microliter, add 295 microliter of water to each sample.
Add then five microliter of RNAase inhibitor concentrated at 40 unit per microliter for a total amount of 200 units per sample and mix gently. Finally add 100 microliter of 5X concentrated IP buffer to each tube. Samples are now ready for the immune precipitation reaction.
Retrieve the magnetic beads coupled with antibody from the rotating wheel and spin them down. Add then 500 microliter of the previously prepared samples to each 100 microliter of beads coupled with the specific antibodies. Mix gently by pipetting up and down several times to completely resuspend the beads.
Incubate them or your meRIP reaction on a rotating wheel at four degrees during two hours. Once incubation is over, spin down each tube and place them on a magnetic rack for one minute to allow bead separation. Then remove the unbound fraction and place it on a fresh tube and keep it for further analysis, if needed.
Remove the magnet from the rack and resuspend all samples in 500 microliter of IP buffer. Resuspend each sample carefully by pipetting up and down. Insert the magnet on the rack, incubate for one minute, and then remove carefully the supernatant.
Repeat the wash twice, and then proceed with elution. For each couple of sample, m6A specific and control, prepare 225 microliter of elution buffer containing 20 millimolar of purified m6A. Then add 100 microliter of elution buffer per sample.
Incubate all sample one hour at four degrees on a rocker. Then collect all m6A enriched RNA sample in a fresh tube and proceed with a second round of elution. Purify the m6A enriched RNA with your method of choice And then at this point samples are ready for library preparation and sequencing.
To investigate the m5C modification, we use bisulfite conversion. Start the experiment with 500 nanogram of poly-adenylated RNA. Although optional, adding to your sample 500 picogram of poly-A depleted RNA to ensure ribosomal representation and 0.5 microliter of commercially available synthetic sequence will allow you to assess bisulfite conversion rate upon sequencing.
Adjust your sample to a final volume of 20 microliter in water, and then add 130 microliter of RNA conversion reagent to each sample. Mix carefully by pipetting up and down and spin down all sample to make sure that there are no droplets left on the side of the tubes. Incubate all sample in a PCR machine for three cycles of denaturation 70 degrees for five minutes and bisulfite conversion at 54 degrees during 45 minutes.
Perform in-column deamination by adding directly on an empty column, 250 microliter of RNA binding buffer, 150 microliter of your bisulfite converted sample. Mix the sample by pipetting up and down, but paying attention in not touching the column filters. Add 400 microliter of 95-100%ethanol to the sample RNA binding buffer mixture directly in the column.
Close the cap and mix immediately by inversion. Spin down your sample for 30 seconds and discard the flow-through. Wash your column with washing buffer and then add 200 microliter of desulphonation solution to the center of each column.
Incubate all your sample for 30 minutes at room temperature. Spin down the sample for 30 seconds and then proceed with two rounds of washing. Add 400 microliter of wash buffer to each sample, centrifuge for 30 seconds at full speed and discard the flow-through.
Place the column into a new empty tube and elute the bisulfite converted RNA into 20 microliter of water. Control bisulfite conversion efficiency by RT-qPCR and then proceed to library preparation and sequencing. Upon bio-informatic analysis, here is some example of results that you may obtain with this workflow.
Here we compare the epitranscriptome of HIV infected cells versus non-infected cells. at 24 hour post-infection. In the A panel, you can see the results we obtain exploring the m6A epitranscriptome.
In this graph, we plot the m6A enriched reads upon meRIP-Seq analysis. We can nicely see a zinc finger is hypermethylated 24 hours post-infection. We performed the same type of analysis for the m5C epitranscriptome investigation in panel B.In this graph, every non-methylated cytosine is displayed in red, while the proportion of cytosine that has been m5C methylated is shown in blue.
As you can see, PHLPP1, all three cytosines that are hyper-methylated 24 hour post-HIV infection. Overall, this workflow allow to investigate both the M6A and m5C epitranscriptome of infected cells and can be applied as such on viral particles as well. Understanding of viral infection are able to modify the host epitranscriptomic landscape will give us access to a new layer of regulation.
The investigation of differential methylated transcripts upon viral infection will provide of course a valuable resource for new therapeutic intervention. At the link here below you can find our open access resource for the investigation of differential methylated transcript upon HIV infection over time.
