3.3 Affinity
Purification
of Polysomes
The following steps take approximately 4 h.
1. Separate the α-FLAG-coupled beads (saved at Subheading 3.2,
step 12) by placing them to a magnet rack for 3 min.
2. Discard the supernatant by pipetting.
3. Use each SN-16 sample (saved at Subheading 3.1, step 7) to
resuspend the beads in one 1.5 mL microfuge tube and transfer
the resuspension to the conical tube containing the rest of the
corresponding SN-16.
4. Incubate for 2 h at 4
C with gentle back-and-forth rocking or
gyration to bind the FLAG-tagged ribosomes to the affinity
beads (see Note 8).
5. Put the tubes in a magnetic rack for 5 min in a 4
C cold room.
6. Transfer the supernatant to a new 15 mL plastic conical tube.
This fraction is the supernatant of the affinity purification or
unbound fraction.
7. Add 6 mL of ice-cold WB to the beads, and mix by gentle backand-forth rocking of the tube. Separate on magnet for 5 min at
4
C and discard supernatant.
8. Repeat 6 mL wash twice as in step 7.
9. Add 1 mL of ice-cold WB and transfer to a new 1.5 mL
microfuge tube at 4
C. Add 200 μL of WB to the original
tube to complete the transfer of the remaining beads.
10. Separate the beads on a small magnetic rack for 3 min. Discard
the supernatant and add 1 mL of ice-cold WB.
11. Repeat 1 mL wash twice as in step 10 and discard the
supernatant.
12. Proceed immediately to RNA extraction (see Note 9).
3.4 RNA Extraction
and Estimation
of RNA Yield
This section takes about 1–2 h. Use a fume hood to work with
TRIzol, chloroform, and isopropanol. After use, TRIzol and chloroform should be discarded as hazardous waste following local
regulations. Optionally, if working with only polyadenylated
RNAs, it is possible to extract those RNAs directly from beads
without the need of a TRIzol extraction (this option is specially
recommended when working with promoters expressed in a low
number of cells). For an example see http://plant-plasticity.github.
io/resources/RNA-prep-magnetic-beads-share.pdf.
1. Process in parallel TRIzol RNA extractions from the TRAP
sample, consisting of washed magnetic beads from Subheading
3.3, step 12, and the SN-16 aliquot put aside in Subheading
3.1, step 8.
TRAP-SEQ Analysis of Long Noncoding RNAs
461
Purification
of Polysomes
The following steps take approximately 4 h.
1. Separate the α-FLAG-coupled beads (saved at Subheading 3.2,
step 12) by placing them to a magnet rack for 3 min.
2. Discard the supernatant by pipetting.
3. Use each SN-16 sample (saved at Subheading 3.1, step 7) to
resuspend the beads in one 1.5 mL microfuge tube and transfer
the resuspension to the conical tube containing the rest of the
corresponding SN-16.
4. Incubate for 2 h at 4
C with gentle back-and-forth rocking or
gyration to bind the FLAG-tagged ribosomes to the affinity
beads (see Note 8).
5. Put the tubes in a magnetic rack for 5 min in a 4
C cold room.
6. Transfer the supernatant to a new 15 mL plastic conical tube.
This fraction is the supernatant of the affinity purification or
unbound fraction.
7. Add 6 mL of ice-cold WB to the beads, and mix by gentle backand-forth rocking of the tube. Separate on magnet for 5 min at
4
C and discard supernatant.
8. Repeat 6 mL wash twice as in step 7.
9. Add 1 mL of ice-cold WB and transfer to a new 1.5 mL
microfuge tube at 4
C. Add 200 μL of WB to the original
tube to complete the transfer of the remaining beads.
10. Separate the beads on a small magnetic rack for 3 min. Discard
the supernatant and add 1 mL of ice-cold WB.
11. Repeat 1 mL wash twice as in step 10 and discard the
supernatant.
12. Proceed immediately to RNA extraction (see Note 9).
3.4 RNA Extraction
and Estimation
of RNA Yield
This section takes about 1–2 h. Use a fume hood to work with
TRIzol, chloroform, and isopropanol. After use, TRIzol and chloroform should be discarded as hazardous waste following local
regulations. Optionally, if working with only polyadenylated
RNAs, it is possible to extract those RNAs directly from beads
without the need of a TRIzol extraction (this option is specially
recommended when working with promoters expressed in a low
number of cells). For an example see http://plant-plasticity.github.
io/resources/RNA-prep-magnetic-beads-share.pdf.
1. Process in parallel TRIzol RNA extractions from the TRAP
sample, consisting of washed magnetic beads from Subheading
3.3, step 12, and the SN-16 aliquot put aside in Subheading
3.1, step 8.
TRAP-SEQ Analysis of Long Noncoding RNAs
461