Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

RNA-binding proteins are essential to cellular physiology. Importantly, the RBP are highly expressed throughout spermatogenesis and have been well-documented as essential post-transcriptional regulators during all stages of germ cells. Identification of binding targets is critical to elucidate the mechanistic roles of RBP.

This protocol represents an adapted application of eCLIP method to capture endogenous direct RNA targets and establishes an applicable basis for eCLIP in mammalian testis. The main advantage of this method is that it is non-radioactive, less time-intensive, and provides a stronger signal-to-noise ratio because the size-matched input will serve as an appropriate background for authentic targets. Lastly, we think that small-scale sequencing of subclones are useful to following deep sequencing.

Demonstrating the procedure will be Xu Qiushi, my colleague. To begin this procedure, harvest about 100 milligrams of testes from mice of appropriate age for each immunoprecipitation experiment. Place the tissues in ice-cold PBS.

Using a pair of fine-tipped tweezers, gently remove the tunica albuginea. Add three milliliters of ice-cold PBS to a tissue grinder, and use a loose glass pestle to triturate the tissue by mild mechanical force. Next, transfer the tissue suspension to a cell culture dish, and add ice-cold PBS up to six milliliters.

Shake the plate quickly so that liquid covers the bottom of the dish evenly. Crosslink the suspension on ice three times with 400 millijoules per centimeter squared at 254 nanometers. Make sure to mix the suspension between each irradiation.

Then, collect the suspension in a 15-milliliter conical tube, and centrifuge at 1, 200 times g and at four degrees for five minutes. Remove the supernatant, and resuspend the pellet in one milliliter of PBS. After this, transfer the suspension to a 1.5-milliliter centrifuge tube.

Centrifuge at 1, 000 times g and at four degrees Celsius for two minutes, and discard the supernatant. First, add 125 microliters of protein A magnetic beads per sample to a fresh centrifuge tube. Place the tube on the magnet to separate the beads from the solution.

After 10 seconds, remove the supernatant, and wash the beads twice with one milliliter of ice-cold lysis buffer. Resuspend the beads in 100 microliters of cold lysis buffer with 10 micrograms of eCLIP antibody. Rotate the tubes at room temperature for 45 minutes.

Then, wash the beads twice with one milliliter of ice-cold lysis buffer. To begin, resuspend the tissue pellets in one milliliter of cold lysis buffer containing 22 microliters of 50x EDTA-free protein inhibitor cocktail and 11 microliters of RNase inhibitor. Keep lysing the samples on ice for 15 minutes.

Sonicate each sample as outlined in the text protocol. Next, add four microliters of DNase to each tube and mix well. Incubate at 37 degrees Celsius for 10 minutes while shaking at 1, 200 rpm.

After this, add 10 microliters of diluted RNase and mix well. Incubate at 37 degrees Celsius for five minutes while shaking at 1, 200 rpm. Then, centrifuge at 15, 000 times g and at four degrees Celsius for 20 minutes to clear the lysate.

Carefully collect the supernatant, and save input samples for RWB and RRI samples. First, add one milliliter of the lysate to the prepared beads, and rotate the samples at four degrees Celsius for either two hours or overnight. After this, collect the beads with a magnetic stand, and discard the supernatant.

Wash the beads twice with 900 microliters of high-salt buffer, and then wash the beads twice with 900 microliters of wash buffer. Then, wash the beads once with 500 microliters of 1x dephosphorylation buffer. First, discard the supernatant, and use fine pipette tips to remove any residual liquid.

Add 25 microliters of three-prime ligation master mix to each sample, and carefully pipette to mix. Add 2.5 microliters of RNA adapter X1A and 2.5 microliters of RNA adapter X1B to each sample. Mix carefully by pipetting or flicking.

Incubate at 25 degrees Celsius for 75 minutes, making sure to flick the tube to mix every 10 minutes. Wash the beads once with 500 microliters of cold wash buffer. Next, wash the beads once with 500 microliters of cold high-salt buffer and then with 500 microliters of cold wash buffer.

Repeat both of these washes once more. Magnetically separate the beads, and use fine pipette tips to remove any residual liquid. Resuspend the beads in 100 microliters of cold wash buffer, and move 20 microliters to new tubes as RWB samples.

Add 7.5 microliters of 4x LDS sample buffer and three microliters of 10x sample reducing agent to the remaining samples. Incubate at 70 degrees Celsius for 10 minutes while shaking at 1, 200 rpm. After this, cool the samples on ice for one minute, and centrifuge at 1, 000 times g and at four degrees Celsius for one minute.

For RWB gel, place tubes on a magnet, and separate protein eluate from the beads. Load 15 microliters of sample into each well. Next, add 500 microliters of antioxidant to 500 milliliters of 1x SDS running buffer.

Run the gel at 200 volts, 1x SDS running buffer for 50 minutes or until the dye front is at the bottom. Transfer the protein-RNA complexes from the gel to a nitrocellulose membrane at 10 volts for 70 minutes in 1x transfer buffer with 10%methanol. Block the RWB membrane in 5%milk in TBST at room temperature for one hour.

Rinse the membrane in TBST, and incubate with primary antibody in TBST overnight at four degrees Celsius. After this, wash twice with TBST, with each wash lasting five minutes. Incubate with secondary antibody in TBST at room temperature for one hour.

Then, wash the membrane three times with TBST. Next, mix equal volumes of ECL buffer A and buffer B.Add this mixture to the membrane, and incubate for one minute. Cover the membrane with plastic wrap, and expose it to an x-ray film at room temperature for two to three minutes before developing the film.

In this study, MOV10 eCLIP in testes from adult wild-type mice with RNase I treating the crosslinked lysate, the target protein, which is approximately 114 kilodaltons, is successfully enriched. The qPCR results of cDNA diluted one to 10 from various samples reveals that non-crosslinked samples show decreased RNA recovery. It is observed that the Ct values of the non-crosslinked group is generally five times more than UV-crosslinked group.

Representative results for PCR amplification and size selection via agarose gel electrophoresis are shown here. The primer-dimer product appears at about 140 base pairs. The UCSC Genome Browser view of two representative subclone sequences shows that MOV10-bound eCLIP tags are found to be located within the three-prime UTR of gene Fto.

The approximate rate of three-prime UTR targets accounts for 75%is consistent with the majority of MOV10 targets in HEK293 cells and in testes. In contrast, MOV10L1-bound eCLIP tags are found to be located within a piRNA cluster, indicating MOV10L1 targets piRNA precursors. The approximate rate of piRNA precursor targets accounts for 42%which reflect a trend from previous studies.

MOV10L1 eCLIP with a 40-unit-per-milliliter RNase I digestion yields relatively more sequences with less than 20 base pairs. This protocol described here represents an employment of the eCLIP method in reproduction, an area in which the RNA-RBP interaction knowledge is rather insufficient.