2 Materials
2.1 Plant Growth
1. Seeds of transgenic plants expressing the RBP of interest fused
to GFP.
2. 50% Solution of commercial bleach: 12% sodium hypochlorite,
0.01% Triton X-100, 70% (v/v) ethanol, and sterile water.
3. Agar plates appropriate for growth of the genotype of interest.
2.2 Fixation
1. Formaldehyde (37% stock solution, fresh).
2. 125 mM Glycine (sterilized by autoclaving).
3. Ice-cold sterile water.
4. Desiccator.
5. Water-jet vacuum pump.
6. 250 mL Erlenmeyer flasks.
7. Absorbent paper tissue.
2.3 Preparation
of Beads
1. Sepharose beads (e.g., IBA).
2. GFP-Trap
® agarose beads (Chromotek).
3. RNA immunoprecipitation lysis buffer (RIP-LB) without inhibitors: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 4 mM
MgCl 2 , 0.25% sodium deoxycholate, 0.25% Igepal CA-630,
1% SDS. Before use, add 5 mM DTT.
4. DNA LoBind
® tubes (Eppendorf).
2.4 Total Extract
Preparation
1. Liquid nitrogen.
2. RiboLock™ (Thermo Fischer Scientific).
3. Vanadylribosyl complex (VRC) RNAse inhibitor (New England Biolabs).
4. Complete
® Protease inhibitor tablets EDTA-free (Roche),
phenylmethylsulfonyl fluoride (PMSF) 1 M in isopropanol.
5. RNA immunoprecipitation lysis buffer (RIP-LB): 50 mM Tris–
HCl, pH 7.5, 150 mM NaCl, 4 mM MgCl 2 , 0.25% Igepal
CA-630. Before use, add 5 mM DTT, 10 mM VRC, 100 U
RiboLock™/mL, 1 mM PMSF, and protease inhibitor tablets
according to the instructions of the supplier (see Note 1).
6. Mortar and pestle.
7. Absorbing paper tissue.
8. Steel balls ø 4 mm.
9. Disposable plastic syringe (10 mL).
10. 0.45 μm Sterile filter.
11. Eppendorf Thermomixer.
RIP-seq
455
2.1 Plant Growth
1. Seeds of transgenic plants expressing the RBP of interest fused
to GFP.
2. 50% Solution of commercial bleach: 12% sodium hypochlorite,
0.01% Triton X-100, 70% (v/v) ethanol, and sterile water.
3. Agar plates appropriate for growth of the genotype of interest.
2.2 Fixation
1. Formaldehyde (37% stock solution, fresh).
2. 125 mM Glycine (sterilized by autoclaving).
3. Ice-cold sterile water.
4. Desiccator.
5. Water-jet vacuum pump.
6. 250 mL Erlenmeyer flasks.
7. Absorbent paper tissue.
2.3 Preparation
of Beads
1. Sepharose beads (e.g., IBA).
2. GFP-Trap
® agarose beads (Chromotek).
3. RNA immunoprecipitation lysis buffer (RIP-LB) without inhibitors: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 4 mM
MgCl 2 , 0.25% sodium deoxycholate, 0.25% Igepal CA-630,
1% SDS. Before use, add 5 mM DTT.
4. DNA LoBind
® tubes (Eppendorf).
2.4 Total Extract
Preparation
1. Liquid nitrogen.
2. RiboLock™ (Thermo Fischer Scientific).
3. Vanadylribosyl complex (VRC) RNAse inhibitor (New England Biolabs).
4. Complete
® Protease inhibitor tablets EDTA-free (Roche),
phenylmethylsulfonyl fluoride (PMSF) 1 M in isopropanol.
5. RNA immunoprecipitation lysis buffer (RIP-LB): 50 mM Tris–
HCl, pH 7.5, 150 mM NaCl, 4 mM MgCl 2 , 0.25% Igepal
CA-630. Before use, add 5 mM DTT, 10 mM VRC, 100 U
RiboLock™/mL, 1 mM PMSF, and protease inhibitor tablets
according to the instructions of the supplier (see Note 1).
6. Mortar and pestle.
7. Absorbing paper tissue.
8. Steel balls ø 4 mm.
9. Disposable plastic syringe (10 mL).
10. 0.45 μm Sterile filter.
11. Eppendorf Thermomixer.
RIP-seq
455