2.5 Immunoprecipitation
1. RNA immunoprecipitation lysis buffer (RIP-LB) (see Subheading 2.4).
2. RNA immunoprecipitation wash buffer (RIP-WB): 50 mM
Tris–HCl, pH 7.5, 500 mM NaCl, 4 mM MgCl 2 , 0.5% Igepal,
0.5% sodium deoxycholate, 1% SDS, 2 M urea. Before use, add
2 mM DTT.
3. End-over-end rotator (e.g., Rotoshake).
4. DNA LoBind
® tubes (Eppendorf).
2.6 RNA Isolation
1. Tri-reagent: 0.8 M guanidinium thiocyanate, 0.4 M ammonium thiocyanate, 4.35% (w/v) glycerol, 0.1 M sodium acetate
pH 5, 38% (v/v) acidic phenol (see Note 2).
2. Chloroform:isoamyl alcohol (24:1).
3. Isopropanol, 70% (v/v) ethanol prechilled at À20
C.
4. Ambion
® GlycoBlue™ (Thermo Fisher Scientific).
5. RQ1 DNase, RQ1 buffer, and RQ1 Stop solution (Promega).
6. 8 M LiCl.
7. Eppendorf Thermomixer.
2.7 Library
Preparation
1. TruSeq
® RNA sample preparation kit v2 (Illumina).
2. Library dilution buffer 10 mM Tris–HCl, pH 8.5, 0.1% (w/v)
Tween 20.
3. Agilent DNA-1000 Kit.
4. Qubit dsRNA HS Assay kit (Life Technologies).
5. Agilent Technologies 2100 Bioanalyzer.
6. Qubit fluorometer (Thermo Fisher Scientific).
3 Methods
3.1 Plant Growth
1. To surface-sterilize Arabidopsis seeds, put seeds into Eppendorf
cap. We plate 100–150 seeds per petri dish (85 mm ø) and use
at least two plates per immunoprecipitation. Add 1 mL of 70%
ethanol, mix, and remove ethanol.
2. Add 1 mL of bleach, mix by inverting several times during
5 min, microcentrifuge at 7500 g, and remove bleach.
3. Wash seeds with sterile water, microcentrifuge at 7500 g,
remove water, and repeat washing twice.
4. Remove water and stratify seeds at 4
C for 2 days in the dark.
5. Germinate and grow plants on agar plates under appropriate
light and temperature conditions.
456
Tino Ko ¨ ster and Dorothee Staiger
1. RNA immunoprecipitation lysis buffer (RIP-LB) (see Subheading 2.4).
2. RNA immunoprecipitation wash buffer (RIP-WB): 50 mM
Tris–HCl, pH 7.5, 500 mM NaCl, 4 mM MgCl 2 , 0.5% Igepal,
0.5% sodium deoxycholate, 1% SDS, 2 M urea. Before use, add
2 mM DTT.
3. End-over-end rotator (e.g., Rotoshake).
4. DNA LoBind
® tubes (Eppendorf).
2.6 RNA Isolation
1. Tri-reagent: 0.8 M guanidinium thiocyanate, 0.4 M ammonium thiocyanate, 4.35% (w/v) glycerol, 0.1 M sodium acetate
pH 5, 38% (v/v) acidic phenol (see Note 2).
2. Chloroform:isoamyl alcohol (24:1).
3. Isopropanol, 70% (v/v) ethanol prechilled at À20
C.
4. Ambion
® GlycoBlue™ (Thermo Fisher Scientific).
5. RQ1 DNase, RQ1 buffer, and RQ1 Stop solution (Promega).
6. 8 M LiCl.
7. Eppendorf Thermomixer.
2.7 Library
Preparation
1. TruSeq
® RNA sample preparation kit v2 (Illumina).
2. Library dilution buffer 10 mM Tris–HCl, pH 8.5, 0.1% (w/v)
Tween 20.
3. Agilent DNA-1000 Kit.
4. Qubit dsRNA HS Assay kit (Life Technologies).
5. Agilent Technologies 2100 Bioanalyzer.
6. Qubit fluorometer (Thermo Fisher Scientific).
3 Methods
3.1 Plant Growth
1. To surface-sterilize Arabidopsis seeds, put seeds into Eppendorf
cap. We plate 100–150 seeds per petri dish (85 mm ø) and use
at least two plates per immunoprecipitation. Add 1 mL of 70%
ethanol, mix, and remove ethanol.
2. Add 1 mL of bleach, mix by inverting several times during
5 min, microcentrifuge at 7500 g, and remove bleach.
3. Wash seeds with sterile water, microcentrifuge at 7500 g,
remove water, and repeat washing twice.
4. Remove water and stratify seeds at 4
C for 2 days in the dark.
5. Germinate and grow plants on agar plates under appropriate
light and temperature conditions.
456
Tino Ko ¨ ster and Dorothee Staiger