With this protocol, we introduced the first method capable of simultaneously profiling multiple RNA modifications in single neurons, opening new areas of investigation involving post-transcriptional regulation in individual cells. By leveraging optimized sample preparation conditions and liquid chromatography tandem mass spectrometry, over a dozen modified nucleosides can be detected and quantified in a single neuron per experiment. Individual cells are different in terms of their functions, morphology, and their chemical profiles.
It's important that we're able to measure the complete chemical mosaic of these cells, including their RNA profiles, for enough cells to understand their differences, both in health and in disease. The extent of ganglia preparation prior to single neuron isolation depends on the preferences of the individual performing the isolation. Attempt both longer and shorter protease treatments to identify suitable conditions.
To begin, place the anesthetized Aplysia californica ventral side up in a dissecting tray. Start dissecting it using surgical scissors with one blunt tip positioned toward the animal and carefully making a longitudinal cut through the foot. After pinning the rostral, caudal, and lateral sides of the animal body to expose the internal organs and the central nervous system ganglia in the body cavity, isolate the major central nervous system ganglia from the animal by surgically severing nerves and some connectives originating from the ganglia.
Immerse the ganglia in 10 milligrams per milliliter protease type XIV solution from Streptomyces griseus and incubate for 30 minutes to one hour at 34 degrees Celsius. Next, rinse the ganglia six times with artificial seawater antibiotic solution. Then using a polypropylene transfer pipette that has been cut to an opening of five millimeter, transfer all ganglia into a silicone polymer-coated dish filled with the artificial seawater antibiotic solution.
Keep the ganglia always submerged in artificial seawater. Next, to remove the ganglion sheaths, pin down the ganglia and use microscissors and fine forceps. With sufficiently strong enzymatic treatment, use glass or metal needles for unsheathing.
Visually identify the neurons of interest. Using a pooled glass capillary or sharp tungsten needles, carefully isolate the identified cell from the bulk ganglion. After drawing about one microliter of artificial seawater into a plastic micropipette, transfer the isolated cell into a PCR sample tube containing four microliters of 0.365 molar ammonium acetate.
For blank measurements, collect five microliter aliquots of the artificial seawater antibiotic solution from the dish containing the ganglion and mix with the digestion buffer. Lyse the isolated neurons by repeated aspiration and dispensing with a micropipette in 0.365 molar ammonium acetate. Some cells may not immediately rupture.
To lyse them, apply pressure across the diameter of the cell with a pooled glass capillary. Next, heat the sample in a thermal cycle before adding 3.375 microliters of RNA digestion buffer to the sample. Mix this solution using a micropipette by withdrawing and dispensing the solution several times.
Spin down any liquid droplets clinging to the walls of the PCR tube using a miniature bench top centrifuge. Then incubate the samples in the thermal cycle at 37 degrees Celsius for three hours, followed by a hold at 10 degrees Celsius with the heated lid set to On.Once the samples have cooled, immediately transfer seven microliters of the solution into an autosampler vial equipped with a 250 microliter insert without any bubble formation in the autosampler tube. To prepare the liquid chromatography system for separation of the canonical and modified nucleosides, equilibrate a C18 column using 99%mobile phase A and 1%mobile phase B at a flow rate of 0.2 milliliters per minute for 12 minutes at 36 degrees Celsius.
Operate the mass spectrometry instrument in positive mode with the diverter valve set to waste for the first two minutes of analysis and to source for the remainder of the run. Then collect mass spectra over an m/z range of 110 to 600. Select ions for collision-induced dissociation at 35 to 40 electron volts over a three second cycle time using a preferred mass list constructed from the database and an isolation window of 0.5.
Use active exclusion to exclude ions from fragmentation after three spectra. Set dynamic MS/MS spectra acquisition for ions with intensities above and below 50, 000 counts at four Hertz and one Hertz, respectively. And a minimum threshold for ion selection at 1, 990 counts.
For quantitative single neuron RNA modification analysis by mass spectrometry, construct calibration curves using an extracted ion chromatogram peak areas obtained for modified nucleoside standards at a minimum of five concentrations to allow interpolation of unknown endogenous analyte concentrations. Single neuron RNA modification analysis by mass spectrometry involves the manual isolation of identified neurons into small sample volumes for lysis digestion and LC-MS/MS analysis. Single neuron RNA modification analysis by mass spectrometry routinely detected over a dozen RNA modifications in single neurons from the central nervous system of Aplysia californica representing coverage of nearly half of the known epitranscriptome of this animal in a single cell.
The results revealed for the first time that RNA modification profiles of single cells could diverge from bulk cells in the same tissue. Further support for unique modified nucleoside patterns was obtained from a separate cohort of animals in which pairwise comparisons were performed for 13 RNA modifications, commonly detected in both the single neurons and bulk tissue. RNA modifications in functionally different cells formed unique clusters in the score plot while homologous R2/LPl1 neurons co-clustered.
The loading plot shows that differences were primarily driven by the abundance of positional isomers of methyladenosine, including 2'O-methyladenosineand and N1-methyladenosine. External calibration curves were generated for N1-methyladenosine, pseudouridine, 2'O-methylguanosine, 2'O-methyladenosine, and N6-dimethyladenosine. And the amount of each modified nucleoside in the metacerebral cells and R2/LPl1 cell pairs was determined by interpolation.
Ensure that a minimum volume of artificial seawater is transferred into the PCR tube along with the single isolated neuron. This minimizes sample dilution, which ultimately affects analyte detection. This protocol can be used for investigating distributions of RNA modifications in subcellular structures, like axons and dendrites, to improve understanding of the mechanisms of activity-dependent local translation.
