13. Use a scalpel and a ruler to cut the desired sizes (15–80 kDa
above the expected molecular weight of the precipitated protein) from the film.
14. Adjust the film to the membrane and use clean scalpels to excise
the desired membrane fragments (see Note 13).
15. Transfer the membrane fragments into 1.5 mL tubes.
3.12 RNA Isolation
1. Add 200 μL of proteinase K buffer and 10 μL proteinase K to
the membrane pieces.
2. Incubate tubes in the thermomixer for 20 min at 37
C and at
1100 rpm.
3. Add 200 μL of PK urea buffer and incubate for another 20 min
at 37
C and 1100 rpm.
4. Collect the supernatant and transfer to a new tube. If necessary,
centrifuge tubes briefly and add remaining solution to the
supernatant sample (see Note 14).
5. Prespin Phase Lock Gel heavy tubes for 30 s at 12,000 Â g to
collect the gel at the bottom of the tubes.
6. Add 400 μL of Tri-reagent to the solution collected from the
membrane.
7. Incubate for 5 min at 30
C and 1100 rpm.
8. Add 400 μL chloroform/isoamyl alcohol (24:1), mix thoroughly by pipetting, and transfer the solution to a 2 mL
Phase Lock tube.
9. Incubate for 5 min at 30
C and 1100 rpm (do not vortex!).
10. Centrifuge for 5 min at 12,000 Â g at room temperature.
11. Transfer the aqueous layer into a new tube. Be careful not to
touch the gel with the pipette.
12. Add 0.8 μL of GlycoBlue and 40 μL 3 M sodium acetate
(pH 5.5) and vortex thoroughly.
13. Add 850 μL of pre-chilled 100% EtOH, mix thoroughly, and
precipitate overnight at À20
C.
14. RNA is ready for preparation of cDNA libraries for highthroughput sequencing as described in [23].
4 Notes
1. Freeze aliquots of the buffer at À20
C and thaw only once.
2. NuPAGE MOPS SDS running buffer is commercially available
(Thermo Fisher Scientific).
3. We recommend to use precast 4–12% NuPAGE Bis-Tris gels as
the NuPAGE buffer system ensures a stable pH around 7.0
Plant iCLIP
265
above the expected molecular weight of the precipitated protein) from the film.
14. Adjust the film to the membrane and use clean scalpels to excise
the desired membrane fragments (see Note 13).
15. Transfer the membrane fragments into 1.5 mL tubes.
3.12 RNA Isolation
1. Add 200 μL of proteinase K buffer and 10 μL proteinase K to
the membrane pieces.
2. Incubate tubes in the thermomixer for 20 min at 37
C and at
1100 rpm.
3. Add 200 μL of PK urea buffer and incubate for another 20 min
at 37
C and 1100 rpm.
4. Collect the supernatant and transfer to a new tube. If necessary,
centrifuge tubes briefly and add remaining solution to the
supernatant sample (see Note 14).
5. Prespin Phase Lock Gel heavy tubes for 30 s at 12,000 Â g to
collect the gel at the bottom of the tubes.
6. Add 400 μL of Tri-reagent to the solution collected from the
membrane.
7. Incubate for 5 min at 30
C and 1100 rpm.
8. Add 400 μL chloroform/isoamyl alcohol (24:1), mix thoroughly by pipetting, and transfer the solution to a 2 mL
Phase Lock tube.
9. Incubate for 5 min at 30
C and 1100 rpm (do not vortex!).
10. Centrifuge for 5 min at 12,000 Â g at room temperature.
11. Transfer the aqueous layer into a new tube. Be careful not to
touch the gel with the pipette.
12. Add 0.8 μL of GlycoBlue and 40 μL 3 M sodium acetate
(pH 5.5) and vortex thoroughly.
13. Add 850 μL of pre-chilled 100% EtOH, mix thoroughly, and
precipitate overnight at À20
C.
14. RNA is ready for preparation of cDNA libraries for highthroughput sequencing as described in [23].
4 Notes
1. Freeze aliquots of the buffer at À20
C and thaw only once.
2. NuPAGE MOPS SDS running buffer is commercially available
(Thermo Fisher Scientific).
3. We recommend to use precast 4–12% NuPAGE Bis-Tris gels as
the NuPAGE buffer system ensures a stable pH around 7.0
Plant iCLIP
265