(see Note 7). Centrifuge for 1 min at 500 Â g. Repeat this
washing step in IP lysis buffer twice. Store at 4
C until use.
3.4 Total Extract
Preparation
1. Remove tubes with frozen plant material from liquid nitrogen
and add 750 μL of IP lysis buffer prewarmed to 40
C (see
Note 8).
2. Add steel ball and replace punctured lid by an intact lid of a new
tube. Invert several times.
3. Incubate briefly at 40
C and 1400 rpm in a thermomixer until
you get a homogenous lysate.
4. Centrifuge for 10 min at maximal speed at 4
C and transfer
supernatant to a fresh 1.5 mL tube. Combine supernatants.
5. Filter lysate using a 5 mL syringe and a 0.45 μm syringe filter.
6. Add 1.0–1.5 mL lysate to the washed sepharose beads and
vortex.
7. Incubate on an end-over-end rotator for 1 h at 4
C.
3.5 Immunoprecipitation
1. Remove any remaining buffer from the GFP-Trap
® beads.
2. Centrifuge precleared samples from Subheading 3.4, step 7,
for 2 min at 500 Â g and 4
C to pellet the sepharose beads.
3. Transfer the precleared lysate to the GFP-Trap
® beads and
incubate on an end-over-end rotator for 1 h at 4
C.
4. Centrifuge for 1 min at 500 Â g at 4
C and discard
supernatants.
5. Wash beads with 850 μL of ice-cold IP wash buffer for 10 min
at 4
C and rotate on an end-over-end rotator (see Note 9).
6. Centrifuge for 1 min at 500 Â g at 4
C and discard
supernatants.
7. Repeat washing three more times.
8. Transfer beads to new LoBind
® tubes.
9. Wash beads twice with 850 μL of ice-cold PNK wash buffer
(pH 7.4) for 1 min at 4
C and rotate on an end-over-end
rotator during each washing step.
10. After each washing step, centrifuge for 1 min at 500 Â g at 4
C
and discard supernatant.
3.6 RNase Digest
1. Resuspend beads in 100 μL PNK wash buffer (pH 7.4).
2. Add 2 μL of Turbo DNase and 5 μL of RNase I dilution (see
Note 10) to the beads and incubate for 10 min in a thermomixer at 37
C and 1100 rpm.
3. Transfer the reaction for more than 3 min on ice.
4. Centrifuge for 2 min at 500 Â g and discard supernatants.
Plant iCLIP
261
washing step in IP lysis buffer twice. Store at 4
C until use.
3.4 Total Extract
Preparation
1. Remove tubes with frozen plant material from liquid nitrogen
and add 750 μL of IP lysis buffer prewarmed to 40
C (see
Note 8).
2. Add steel ball and replace punctured lid by an intact lid of a new
tube. Invert several times.
3. Incubate briefly at 40
C and 1400 rpm in a thermomixer until
you get a homogenous lysate.
4. Centrifuge for 10 min at maximal speed at 4
C and transfer
supernatant to a fresh 1.5 mL tube. Combine supernatants.
5. Filter lysate using a 5 mL syringe and a 0.45 μm syringe filter.
6. Add 1.0–1.5 mL lysate to the washed sepharose beads and
vortex.
7. Incubate on an end-over-end rotator for 1 h at 4
C.
3.5 Immunoprecipitation
1. Remove any remaining buffer from the GFP-Trap
® beads.
2. Centrifuge precleared samples from Subheading 3.4, step 7,
for 2 min at 500 Â g and 4
C to pellet the sepharose beads.
3. Transfer the precleared lysate to the GFP-Trap
® beads and
incubate on an end-over-end rotator for 1 h at 4
C.
4. Centrifuge for 1 min at 500 Â g at 4
C and discard
supernatants.
5. Wash beads with 850 μL of ice-cold IP wash buffer for 10 min
at 4
C and rotate on an end-over-end rotator (see Note 9).
6. Centrifuge for 1 min at 500 Â g at 4
C and discard
supernatants.
7. Repeat washing three more times.
8. Transfer beads to new LoBind
® tubes.
9. Wash beads twice with 850 μL of ice-cold PNK wash buffer
(pH 7.4) for 1 min at 4
C and rotate on an end-over-end
rotator during each washing step.
10. After each washing step, centrifuge for 1 min at 500 Â g at 4
C
and discard supernatant.
3.6 RNase Digest
1. Resuspend beads in 100 μL PNK wash buffer (pH 7.4).
2. Add 2 μL of Turbo DNase and 5 μL of RNase I dilution (see
Note 10) to the beads and incubate for 10 min in a thermomixer at 37
C and 1100 rpm.
3. Transfer the reaction for more than 3 min on ice.
4. Centrifuge for 2 min at 500 Â g and discard supernatants.
Plant iCLIP
261