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

Reagents

Product No.	Description
RIP	Imprint® RNA Immunoprecipitation Kit
SAB4200085	Monoclonal Anti-AGO2 antibody produced in rat, clone 11A9
R9255	Anti-Rat IgG (whole molecule) antibody produced in rabbit (aka, rabbit anti-rat)
I4131	IgG from rat serum
09691	Ammonium acetate solution, 5M
Ambion AM9520	Linear Acrylamide (5 mg/mL)
I9516	2-Propanol

Reagents for Harsh/Stringent Wash Solution

I8896	IGEPAL® CA-630
S5150	Sodium chloride solution 5M

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

Reagent	per RIP	2.1 RIPs
Lysis buffer	0.2	0.42 mL
PIC (protease inhibitor cocktail)	2.0	4.2 µL
RNase inhibitor	0.8	1.68 µL

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:

Reagent	Proportions	2.1 x mild	2.1 x harsh
Wash buffer	1	0.63 mL	1.68 mL
PIC (protease inhibitor cocktail)	10	6.3 µL	16.8 µL
RNase inhibitor	4	2.52 µL	6.72 µL

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

Stringent Wash Buffer

Reagent	Initial	Final	10 mL
Wash buffer		1	10 mL
100% Igepal	0.05	1	0.095 mL
5 M NaCl	0.15	0.9	1.5 mL

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

Reagents

Imprint RNA Immunoprecipitation Kit	RIP
RIP Kit components required:
Mild Lysis Buffer	B0314
Harsh Lysis Buffer	B0439
RIP Wash Buffer	B0564
Protein A Magnetic Beads	B0689
Ribonuclease Inhibitor	R1158
Protease Inhibitor Cocktail	P8340

Additional Reagents

Monoclonal Anti-AGO2 antibody produced in rat, clone 11A9	SAB4200085
Anti-Rat IgG (whole molecule) antibody produced in rabbit	R9255
IgG from rat serum	I4131
TRI Reagent	T9424
Chloroform	C2432
Ammonium acetate solution, 5M	09691
2-Propanol	I9516
Linear Acrylamide (5 mg/mL)	Ambion AM9520

Reagents for Making Harsh/Stringent Wash Solution

IGEPAL CA-630	I8896
Sodium chloride solution, 5M	S5150