RIP-Seq Sample Preparation Protocol

Obtain Cell Lysate

1. Wash the cells in the culture dish or culture flask twice with cold PBS.
2. Add cold PBS and scrape down the cells with a cell scraper and collect them into eppendoff tubes.
3. Centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant and collect the cells.
4. Resuspend the cells with the same volume of RIP lysate as the cells, blow well and leave on ice for 5 min.
5. Dispense 200 μl of cell lysate in each tube and store at -80°C.

Preparation of magnetic beads and antibodies

1. Resuspension of magnetic beads.
2. Label the eppendoff tubes required for the experiment.
3. Pipette 50 μl of the resuspended beads into each eppendoff tube.
4. Add 500 μl of RIP Wash Buffer to each tube and vortex shake.
5. Place eppendoff tubes on a magnetic stand, remove supernatant, repeat once.
6. Resuspend beads with 100 μl of RIP Wash Buffer.
7. Incubate at room temperature for 30 min.
8. Place the eppendoff tube on a magnetic stand and discard the supernatant.
9. Add 500 μl RIP Wash Buffer, vortex and shake, then discard supernatant, repeat once.
10. Add 500 μl RIP Wash Buffer, vortex and shake, then place on ice.

RNA-Binding Protein Immunoprecipitation

1. Prepare RIP Immunoprecipitation Buffer.
2. Place the eppendoff tubes from the previous step on a magnetic rack, remove the supernatant, and add 900 ul of RIP Immunoprecipitation Buffer to each tube.
3. Quickly thaw the cell lysate prepared in the first step and centrifuge. Aspirate 100 μl of supernatant in the magnetic bead-antibody complex from the previous step to make a total volume of 1 ml.
4. Incubate for 3 h at 4°C until overnight.
5. Centrifuge briefly, place the eppendoff tube on a magnetic stand and discard the supernatant.
6. Add 500 μl RIP Wash Buffer, vortex and shake the eppendoff tube on the magnetic rack, discard the supernatant, repeat washing 6 times.

RNA Purification

1. Prepare Proteinase K Buffer. 150 ul for each sample.
2. Resuspend the above magnetic beads-antibody complex with 150 ul Proteinase K Buffer.
3. Incubate at 55°C for 30min.
4. After incubation, place the enpendoff tube on a magnetic stand and draw the supernatant into a new enpendoff tube.
5. Add 250 μl of RIP Wash Buffer to each supernatant tube.
6. Add 400 μl of phenol:chloroform:isoamyl alcohol to each tube, vortex and shake, and centrifuge at 14,000 rpm for 10 min at room temperature.
7. Carefully aspirate 350 μl of the upper aqueous phase into a new enpendoff tube.
8. Add 400 μl of chloroform to each tube, vortex shake for 15 s, and centrifuge at 14,000rpm for 10min at room temperature
9. Carefully aspirate 300 μl of the upper aqueous layer into a new enpendoff tube.
10. Add 50 μl Solution I, 15 μl Solution II, 5 μl of Precipitate Enhancer, 850 μl of anhydrous ethanol (without RNase) to each tube, mix, and keep at -80°C for 1h until overnight.
11. Centrifuge at 14,000 rpm, 4°C for 30 min, carefully remove supernatant.
12. Rinse once with 80% ethanol, centrifuge at 14,000 rpm for 15 min at 4°C, carefully remove supernatant and air dry.
13. Dissolve 10-20 μl DEPC in water and store at -80℃.