1. Transfer 5 μL of 100 μM stock of RNA probe to a new 2 mL
tube (see Note 9).
2. Add 35 μL of RNase-free water.
3. Add 360 μL of 5 mM sodium m-periodate in 0.1 M NaOAc (see
Note 10).
4. Incubate for 1 h in the dark at room temperature (see Note 11).
5. Add 3 μL of 5 mg/mL glycogen, 23 μL 3 M NaOAc, and 1 mL
100% EtOH.
6. Shake vigorously and then centrifuge down.
7. Store at À80
C > 2 h (see Note 12).
8. Centrifuge at max speed for >80 min at 4
C (see Note 13).
9. Aspirate supernatant.
10. Wash with 750 μL 80% ethanol.
11. Spin for 5 min at max speed at room temperature in a microcentrifuge, remove supernatant, and let air-dry until only the
pellet remains (see Note 14).
12. During ethanol washing (step 11), prepare and label new 2 mL
tubes.
13. Resuspend pellet in 290 μL RNase-free water and transfer the
content to a new tube.
14. Add 10 μL 3 M NaOAc for a final volume and concentration of
300 μL in 0.1 M NaOAc.
15. During centrifuging step (step 8), transfer 100 μL of adipic
acid dihydrazide agarose beads in 50% solution to a 15 mL
tube, one for each sample (see Note 15).
16. Wash 4Â with 10 mL 0.1 M NaOAc; each spin is at 1439 rcf
for 5 min.
17. Resuspend beads in 900 μL/probe 0.1 M NaOAc (see Note
16).
18. Add beads to the RNA, cover each tube with tinfoil, and let
rotate at 4
C overnight.
If possible, calculate and make up enough of the buffers for
day 2. Buffers can be stored for short term at room temperature.
3.3.2 Incubating Tagged
RNA with Protein Lysate
During the previous day, RNA probes were tagged with agarose
beads. At the successful completion of the following steps, the
tagged RNAs will be incubated with protein lysates to encourage
the binding of RBPs to the RNA sequences of interest. The various
washes will remove nonspecific protein binding before the reaction
is digested with trypsin overnight to fragment the bound proteins
into peptide fragments.
248
Mengge Shan and Brian D. Gregory
tube (see Note 9).
2. Add 35 μL of RNase-free water.
3. Add 360 μL of 5 mM sodium m-periodate in 0.1 M NaOAc (see
Note 10).
4. Incubate for 1 h in the dark at room temperature (see Note 11).
5. Add 3 μL of 5 mg/mL glycogen, 23 μL 3 M NaOAc, and 1 mL
100% EtOH.
6. Shake vigorously and then centrifuge down.
7. Store at À80
C > 2 h (see Note 12).
8. Centrifuge at max speed for >80 min at 4
C (see Note 13).
9. Aspirate supernatant.
10. Wash with 750 μL 80% ethanol.
11. Spin for 5 min at max speed at room temperature in a microcentrifuge, remove supernatant, and let air-dry until only the
pellet remains (see Note 14).
12. During ethanol washing (step 11), prepare and label new 2 mL
tubes.
13. Resuspend pellet in 290 μL RNase-free water and transfer the
content to a new tube.
14. Add 10 μL 3 M NaOAc for a final volume and concentration of
300 μL in 0.1 M NaOAc.
15. During centrifuging step (step 8), transfer 100 μL of adipic
acid dihydrazide agarose beads in 50% solution to a 15 mL
tube, one for each sample (see Note 15).
16. Wash 4Â with 10 mL 0.1 M NaOAc; each spin is at 1439 rcf
for 5 min.
17. Resuspend beads in 900 μL/probe 0.1 M NaOAc (see Note
16).
18. Add beads to the RNA, cover each tube with tinfoil, and let
rotate at 4
C overnight.
If possible, calculate and make up enough of the buffers for
day 2. Buffers can be stored for short term at room temperature.
3.3.2 Incubating Tagged
RNA with Protein Lysate
During the previous day, RNA probes were tagged with agarose
beads. At the successful completion of the following steps, the
tagged RNAs will be incubated with protein lysates to encourage
the binding of RBPs to the RNA sequences of interest. The various
washes will remove nonspecific protein binding before the reaction
is digested with trypsin overnight to fragment the bound proteins
into peptide fragments.
248
Mengge Shan and Brian D. Gregory