实验步骤
1. UV cross-linking of tissue culture cells
1) Remove media and add ice-cold PBS to cells (e.g. use cells grown in a 10 cm plate for three experiments and add 6 ml PBS)
2) Remove lid, place on ice and irradiate once with 150 mJ/cm2 at 254 nm using a stratalinker
*One or more negative controls should be maintained throughout the complete experiment. Knockout cells or tissue as well as non-cross-linked cells are good negative controls, while knockdown cells are not recommended.
3) Harvest cells with a cell scraper and transfer cell suspension to microtubes (e.g. 2 ml to each of three microtubes)
4) Pellet cells (spin at top speed for 10 sec at 4°C), then remove supernatant
5) Snap-freeze cell pellets on dry ice and store at -80°C until use
*Experiment could take up to a week. Avoid multiple cycles of freeze thaw
2. Bead preparation
1) Add protein A (or protein G) beads (e.g. 100 μl magnetic beads per experiment) to a fresh microtube (use protein G beads for mouse or goat antibodies) and wash beads 2x with lysis buffer
2) Resuspend beads in lysis buffer (100 μl), add antibody (2-10 μg) and rotate tubes at room temperature for 30-60 min
*The amount of antibody required might need to be optimised. A no-antibody sample is a good negative control.
3) Wash beads 3x with lysis buffer (900 μl) and leave in the last wash until ready to proceed with the immunoprecipitation (step 4.1)
*If an antibody is working in IP, this is a good indication that it will work in CLIP.
3. Cell lysis and partial RNA digestion
1) Resuspend cell pellet in lysis buffer (1 ml) and transfer to 1.5 ml microtubes
2) Add low RNase dilution (10 μl) and Turbo DNase (2 μl) to the cell lysate and incubate for exactly 3 min at 37°C, shaking at 1,100 rpm, then immediately transfer to ice
3) Spin at 4°C at 22,000 g for 20 min and carefully collect the cleared supernatant (leave about 50 μl lysate with the pellet)
*Each member of the laboratory should use their own set of buffers and reagents to easier identify potential
sources of contamination. Ideal conditions for the RNase digestions may need to be optimized for every new batch of RNase.
4. Immunoprecipitation and dephosphorylation of RNA 3'ends
1) Remove lysis buffer from the beads (step 2.3) and add cell lysate (from step 3.3)
2) Rotate the samples for 2 h at 4°C
3) Discard the supernatant, wash beads 2x with high-salt buffer (900 μl) and then 2x with wash buffer (900 μl)
*Optimization and stringent washing conditions are very important.
4) Discard the supernatant, resuspend beads in PNK mix (20 μl) and incubate for 20 min at 37°C
5) Add wash buffer (500 μl), wash 1x with high-salt buffer and then 2x with wash buffer
5. Linker ligation to RNA 3' ends and RNA 5' end labelling
1) Carefully remove the supernatant, resuspend beads in ligation mix A (20 μl) and incubate overnight at 16°C
2) Add wash buffer (500 μl), wash 2x with high-salt buffer (1 ml) and then 2x with wash buffer (1 ml)
3) Remove the supernatant, resuspend beads in hot PNK mix (8 μl) and incubate for 5 min at 37°C
4) Remove the hot PNK mix and resuspend beads in 1x SDS-PAGE loading buffer (20 μl)
5) Incubate on a thermomixer at 70°C for 10 min
6) Immediately place on a magnet to precipitate the empty beads and load the supernatant on the gel (see step 5)
6. SDS-PAGE and membrane transfer
1) Load samples as well as a pre-stained protein size marker (5 μl) on a precast 4-12% Bis-Tris gel, and run the gel for 50 min at 180 V in 1x MOPS running buffer (according to manufacturer's instructions)
*Gels with constant pH 7 are recommended.
2) Remove the gel front and discard as solid waste (contains free radioactive ATP)
3) Transfer the protein-RNA complexes from the gel to a nitrocellulose membrane using a wet transfer apparatus (transfer 1 h at 30 V depending on manufacturer's instructions)
*More information on SDS-PAGE and transfer can be found under our Western blot protocols.
4) Following the transfer, rinse the membrane in PBS buffer, then wrap it in clingfilm and expose it to a film at -80°C for 30 min, 1h and then over night
*A fluorescent sticker next to the membrane later allows to align the
film and the membrane. The success of the experiment can be monitored
at the autoradiograph of the protein-RNA complex after membrane transfer
*Control experiments should give no signal on autoradiograph. In the autoradiograph of the low-RNase samples, diffuse radioactivity should be seen above the molecular weight of the protein. For high-RNase samples, this radioactivity is focused closer to the molecular weight of the protein.
7. RNA isolation
1) Isolate the protein-RNA complexes from the membrane using the autoradiograph from step 6.4 as a mask. Cut this piece of membrane into several small slices and place them into a 1.5 ml microtube
2) Add PK buffer (200 μl) and proteinase K (10 μl) to the membrane pieces, and incubate shaking at 1,100 rpm for 20 min at 37°C
3) Add PKurea buffer (200 μl) and incubate for 20 min at 37°C
4) Collect the solution, add it together with RNA phenol/chloroform (400 μl) to a 2 ml Phase Lock Gel Heavy tube and incubate shaking at 1,100 rpm for 5 min at 30°C
5) Separate the phases by spinning for 5 min at 13,000 rpm at room temperature
6) Carefully transfer just the aqueous layer into a new tube
7) Add glycoblue (0.5 μl) and 3 M sodium acetate pH 5.5 (40 μl) and mix. Then add 100% ethanol (1 ml), mix again and precipitate over night at -20°C
8. Reverse transcription
1) Spin for 20 min at 15,000 rpm at 4°C, then remove the supernatant and wash the pellet with 80% ethanol (0.5 ml)
2) Resuspend the pellet in RNA/primer mix (7.25 μl). For each experiment or replicate, use a different Rclip primer containing individual barcode sequences (see 12)
3) Incubate for 5 min at 70°C before cooling to 25°C
4) Add RT mix (2.75 μl) and incubate 5 min at 25°C, 20 min at 42°C, 40 min at 50°C and 5 min at 80°C before cooling to 4°C
5) Add TE buffer (90 μl), glycoblue (0.5 μl) and sodium acetate pH 5.5 (10 μl) and mix; then add 100% ethanol (250 μl), mix again and precipitate over night at -20°C
首页
