Sepharose beads. Mix by vortexing. Incubate on an end-overend rotator for 1–2 h at 4
C.
2. Microcentrifuge precleared samples for 2 min at 500 g and
4
C, to remove Sepharose beads.
3. Transfer supernatant of precleared samples to Eppendorf caps
with washed GFP-Trap
® beads (see step 2 in Subheading 3.3)
for immunoprecipitation (see Note 7).
4. Incubate samples on an end-over-end rotator for 1 h at 4
C.
5. Microcentrifuge for 1 min at 500 g.
6. Transfer supernatants to new Eppendorf caps and freeze at
À20
C.
7. Wash beads with 1 mL of ice-cold RIP-WB at 4
C and microcentrifuge for 1 min at 500 g (see Note 8).
8. Repeat washing (step 7) four more times.
9. Wash beads with 1 mL of ice-cold RIP-LB at 4
C.
3.6 RNA Isolation
1. RNA is purified from the immunoprecipitated RNPs using the
Trizol procedure (see Note 9). Add 400 μL of TriReagent to
the GFP-Trap
® beads.
2. Vortex for 15 s.
3. Incubate for 5 min at 65
C and 1400 rpm in an Eppendorf
Thermomixer (see Note 10).
4. Add 100 μL of chloroform:isoamyl alcohol, mix vigorously for
3 min, and incubate for a further 3 min at room temperature.
5. Microcentrifuge at 12,000 g and 4
C for 15 min. Carefully
remove upper phase and transfer to new Eppendorf cap.
6. Add 1 μL of GlycoBlue™ and 400 μL isopropanol, mix, and
incubate for 30–45 min at room temperature. GlycoBlue™
acts as a carrier to enhance the recovery and improves the
visibility of the RNA pellet.
7. Microcentrifuge at 12,000 g at 4
C for 10 min and discard
supernatant.
8. Wash RNA pellets with ethanol prechilled at À20
C and
microcentrifuge at 12,000 g at 4
C for 5 min (see Note 11).
9. Repeat washing step.
10. Air-dry RNA pellets and resuspend in 16 μL RNase-free water
(see Note 12).
11. Add 2 μL of RQ1 buffer and 2 μL RQ1 DNase to 16 μL of
RNA samples. Mix by flicking the tube or by pipetting up and
down. Incubate for 45 min at 37
C.
12. Add 2 μL of RQ1 stop solution and incubate for 10 min at
65
C.
458
Tino Ko ¨ ster and Dorothee Staiger
C.
2. Microcentrifuge precleared samples for 2 min at 500 g and
4
C, to remove Sepharose beads.
3. Transfer supernatant of precleared samples to Eppendorf caps
with washed GFP-Trap
® beads (see step 2 in Subheading 3.3)
for immunoprecipitation (see Note 7).
4. Incubate samples on an end-over-end rotator for 1 h at 4
C.
5. Microcentrifuge for 1 min at 500 g.
6. Transfer supernatants to new Eppendorf caps and freeze at
À20
C.
7. Wash beads with 1 mL of ice-cold RIP-WB at 4
C and microcentrifuge for 1 min at 500 g (see Note 8).
8. Repeat washing (step 7) four more times.
9. Wash beads with 1 mL of ice-cold RIP-LB at 4
C.
3.6 RNA Isolation
1. RNA is purified from the immunoprecipitated RNPs using the
Trizol procedure (see Note 9). Add 400 μL of TriReagent to
the GFP-Trap
® beads.
2. Vortex for 15 s.
3. Incubate for 5 min at 65
C and 1400 rpm in an Eppendorf
Thermomixer (see Note 10).
4. Add 100 μL of chloroform:isoamyl alcohol, mix vigorously for
3 min, and incubate for a further 3 min at room temperature.
5. Microcentrifuge at 12,000 g and 4
C for 15 min. Carefully
remove upper phase and transfer to new Eppendorf cap.
6. Add 1 μL of GlycoBlue™ and 400 μL isopropanol, mix, and
incubate for 30–45 min at room temperature. GlycoBlue™
acts as a carrier to enhance the recovery and improves the
visibility of the RNA pellet.
7. Microcentrifuge at 12,000 g at 4
C for 10 min and discard
supernatant.
8. Wash RNA pellets with ethanol prechilled at À20
C and
microcentrifuge at 12,000 g at 4
C for 5 min (see Note 11).
9. Repeat washing step.
10. Air-dry RNA pellets and resuspend in 16 μL RNase-free water
(see Note 12).
11. Add 2 μL of RQ1 buffer and 2 μL RQ1 DNase to 16 μL of
RNA samples. Mix by flicking the tube or by pipetting up and
down. Incubate for 45 min at 37
C.
12. Add 2 μL of RQ1 stop solution and incubate for 10 min at
65
C.
458
Tino Ko ¨ ster and Dorothee Staiger