Protocol for Anti Ago-RNA Immunoprecipitation from Mammalian Cells Using the RIP Kit

Prep Cells

1. Grow cells to ~80% confluency. You will need ~2 million cells/RIP, or ~4 million cells for IgG (neg control) & Ago2 RIP
2. Rinse cells with HBSS (volume = culture medium)
3. If cells attached, release in trypsin-EDTA ~5 min and quench with 4-5 volumes serum-containing medium
4. Count cells, aliquot 2 million cells/ tube, and pellet at 4 ^oC, 1000 rpm, 5 min. Discard supernatant.
5. Wash 2x with ice cold PBS (volume = medium removed).
6. Discard supernatant & add 200 µL mild or harsh lysis buffer + Protease Inhibitor Cocktail (PIC) (P8340) + Ribonuclease Inhibitor (RNase inhibitor)(R1158)/cell pellet

7. Incubate on ice 15' (or store o/n at -80 ^oC)
8. Pellet debris at 4 ^oC, 16,000g, 10 min
9. Transfer sup to fresh tube
10. Remove 10 µL from each cell lysate for 5% input and store on ice

Prep Beads

1. Add 40 µL Protein A Magnetic Beads to 200µL wash buffer in 1.5mL tube for 2 RIPs (IgG & Ago2)
2. Place tubes on magnetic stand & remove liquid
3. Wash 1x 200 µL wash buffer
4. Resuspend in 200 µL wash buffer
5. Add 5 µg rabbit Anti-Rat IgG (whole molecule) antibody produced in rabbit (R9255)
6. Incubate at RT with rotation for 30 min.
7. Spin briefly & remove liquid on magnetic stand
8. Wash 2x with 1 mL wash buffer.
9. Resuspend in 200 µL wash buffer & split into 2 x 100 µL
10. Add 2.5 µg rat IgG (I4131) or Monoclonal Anti-AGO2 antibody produced in rat, clone 11A9 (SAB4200085).
11. Incubate at RT with rotation for 30 min.
12. Spin briefly & remove liquid on magnetic stand.
13. Wash 2 times with 0.5 mL wash buffer.
14. Remove wash buffer completely on magnetic stand.

Doing RIP

1. Prep IP Buffer:

1. Resuspend bead pellets in 0.2 mL IP Buffer
2. Verify 5% input reserved from each cell lysate and stored on ice
3. Add 0.1 mL IP Buffer/cells in mild lysis or 0.6 mL/cells in harsh lysis
4. Transfer diluted lysate to resuspended beads
5. Incubate at 4 ^oC with rotation O/N

Wash

1a. For mild washing, wash 5x by adding 1mL wash buffer, vortex, spin briefly, collect beads on magnet, & remove
supernatent
1b. For harsh or stringent washing, wash 1x 1ml wash buffer, 2x 1mL stringent wash buffer, and 2x 1mL standard wash buffer

2. After last wash, resuspend in 0.2 mL wash buffer
3. Bring reserved input samples to 0.2 mL with wash buffer

Purify RNA

1. Add 0.5 mL Tri Reagent (T9424) to each 0.2 mL RIP or inpu
2. Add 0.1 mL chloroform (C2432) to each, vortex thoroughly, and spin at 4 ^oC, 16,000g, 10 min
3. Prepare 1.5 mL tubes containing
   6 µL linear acrylamide (Ambion; 5mg/mL)
   60 µL 5M ammonium acetate (09691)
   600 µL 2-propanol (I9516)
4. Transfer aqueous to tubes from above, vortex, & precipitate at -80 ^oC at least 1 hour
5. Thaw tubes from -80 ^oC on ice
6. Spin at 4 ^oC, 16,000g, 10 min.
7. Carefully pour off supernatant
8. Wash once with 0.5 mL 70% ethanol
9. Spin at 4 ^oC, 16,000g, 10 min.
10. Carefully pour off sup and dry pellets in a laminar flow hood
11. Rehydrate each in 10 µL RNase-free water