The overall goal of this video is to describe the method for sequencing of psoralen crosslinked ligated and selected hybrids or SPLASH in which pair-wise in vivo RNA-RNA interactions are mapped in a genome-wide manner. This technique is a simple, high throughput method for mapping the RNA interaction in vivo. This method can help answer key questions in the RNA genomics field such as how do different regions along a single strand of RNA or between different strands of RNAs interact with each other.
Though this method can provide insights into yeast and human cells, it can also be applied to studies in other modern organisms including bacteria and membrane in cells. We first had the idea for this method when we wanted to come up with a way to map interaction molecular. Note that the following procedure contains several stopping points.
At these times, the experiment can be paused briefly or indefinitely. Details can be found in the accompanying text. Begin this procedure by washing HeLa cells twice with five milliliters of PBS.
Place the dish vertically for one minute to completely drain any excess PBS. Next, add one milliliter of PBS containing 200 micromolar biotinylated psoralen and 0.01%digitonin which increases cell permeability. Take care to add the solution evenly over the surface of the cells.
Incubate at 37 degrees Celsius for five minutes. Following the incubation, remove the lid of the dish and place it on ice inside a UV Crosslinker three centimeters from the UV bulb. Irradiate with 365 nanometer UV for 20 minutes.
After 20 minutes, remove the dish from the Crosslinker and then isolate fragment and precipitate the RNA from HeLa cells according to the instructions in the accompanying document. Load denatured ladder and RNA samples onto a 8.6 centimeter square 6%TBE-urea gel. Size fractionate by electrophoresis at 180 volts for 40 minutes.
Stain the gel in 10 milliliters of TBE buffer containing a 1:10, 000 dilution of nucleic acid gel stain for five minutes in the dark. While the gel is staining, use a 24 gauge needle to puncture a clean 0.6 milliliter microfuge tube at the bottom. Place it in a two milliliter microfuge tube.
Using a transilluminator, visualize the bands on the post-stained gel and cut a gel slice corresponding to 90 to 110 nucleotides. Transfer the gel slice into the punctured 0.6 milliliter microfuge tube in the two milliliter microfuge tube. Size selection allows us to set the minimal length for the chimeric fragments and to distinguish between ligated products and non-ligated products later on.
Centrifuge the gel slices at 12, 000 times g at room temperature for two minutes to shred the gel slice and collect it in the two milliliter microfuge tube. After the spin, discard the empty 0.6 milliliter microfuge tube. To the shredded gel slice, add 700 microliters of elution buffer.
Incubate at four degrees Celsius overnight with constant rotation to allow diffusion of the samples into the buffer. Transfer the gel slices and the elution buffer to a centrifuge tube filter and centrifuge at 20, 000 times g for 20 minutes. After the spin, the gel slices will be trapped at the top compartment of the filter which may be discarded.
Precipitate and quantify the RNA as described in the accompanying document. Flash freeze and store the RNA at minus 80 degrees Celsius in liquid nitrogen until it is used. To enrich RNA crosslinking regions, begin by adding 100 microliters of lysis buffer containing RNase inhibitor to wash streptavidin-coated magnetic beads in a microfuge tube.
To a 15 milliliter conical centrifuge tube, add two milliliters of freshly-prepared hybridization buffer, one milliliter of supplemented lysis buffer, 1.5 micrograms of size fractionated RNA, and 100 microliters of resuspended beads. Vortex the tube gently. Incubate at 37 degrees Celsius for 30 minutes with end-to-end rotation.
Following the incubation, wash the beads five times with pre-warmed wash buffer. At the end of the fifth wash, place the microfuge tube containing the beads on the magnet stand for one minute and then remove the wash buffer. Add one milliliter of cold T4 polynucleotide kinase buffer to the washed beads.
Incubate the beads for five minutes at four degrees Celsius with end-to-end rotation. Place the microfuge tube containing the beads on the magnetic strip. After one minute, gently remove the buffer.
Repeat this step for a total of two washes and remove the buffer after the last wash. Next, follow the instructions in the accompanying document to convert the five prime and three prime ends to be ligation compatible and perform the proximity ligation. After a wash, add 100 microliters of RNA PK buffer to the beads and resuspend them by pipetting up and down.
Heat the samples at 95 degrees Celsius for 10 minutes on a heat block. Next, chill the sample on ice for one minute. Add 500 microliters of guanidinium thiocyanate-phenol-chloroform to the sample.
Mix by vortexing vigorously for 10 seconds. Incubate the mixture at room temperature for 10 minutes. Following the incubation, use a kit to precipitate, clean up, and recover the RNA, ensure that even small RNAs are retained.
Elute the RNA in 100 microliters of nuclease-free water. Transfer 100 microliters of the eluted samples into a well of a 24-well plate on nice. Keeping the sample on ice, UV irradiate it at 254 nanometers for five minutes.
Transfer the reverse crosslinked sample to a clean microfuge tube. Add 10 microliters of sodium acetate, one microliter of glycogen, and 300 microliters of 100%ethanol. Precipitate the RNA at minus 20 degrees Celsius for at least one hour.
To recover the RNA, centrifuge the precipitated RNA at 20, 000 times g for 30 minutes at four degrees Celsius. After the spin, remove the supernatant and add one milliliter of 70%cold ethanol to wash the RNA pellet. Centrifuge the washed pellet at 20, 000 times g for 15 minutes at four degrees Celsius.
Remove the wash buffer and add 4.25 microliters of nuclease-free water to resuspend the RNA. Transfer the RNA to a 0.2 milliliter PCR tube. Finally, reverse transcribe, circularize, and amplify the cDNA according to the instructions in the accompanying document.
This schematic depicts the SPLASH workflow. Upon the addition of biotinylated psoralen in the presence of 0.01%digitonin and UV crosslinking, total RNA is extracted from the cells and a dot blot is performed to ensure that crosslinking was efficient. Biotinylated 20 base oligos are then used as positive controls to titrate the amount of biotinylated psoralen to be added to the cells such that approximately one in every 150 bases are crosslinked.
Small-scale PCR amplification is then performed using increasing numbers of PCR cycles to determine the minium number of cycles required for deep sequencing. In an efficient library preparation process, a cDNA sequencing library can be amplified from a 1.5 microgram of size selected RNA input in less than 15 cycles of PCR amplification. The library is then sequenced using two 150 base pair reads on a high throughout sequencing machine.
The sequencing reads are then processed according to the computational pipeline. The end result is a list of filtered chimeric interactions that includes both intramolecular and intermolecular RNA-RNA interactions in the transcriptome. While attempting this procedure, it is important to remember to check for biotinylated psoralen incorporation when using SPLASH on new organisms.
After its development, this technique paved the way for researchers in the field of RNA biology to explore RNA interactions in diverse organisms. After watching this video, you should have a good understanding of how to make a sequencing library that captures pair-wise RNA interactions globally in the cell.
