RNA Detection

10. Place the tube containing lysate and the beads on the magnet
    and transfer the supernatant to a prechilled new tube on ice.

3.4
Immunoprecipitation
and RNA Purification

1. Add 2.5μL Rabbit anti-HA antibody to the precleared lysate
   (step 10of Subheading3.3) and rotate the tube (head-to-toe
   rotation) in cold room overnight.


2. Wash 40μL Dynabeads with CHX-lysis buffer assteps 5– 7 of
   the Subheading3.3, and add it to the lysate containing the
   antibody (step 1). Do not let the beads dry out.


3. After 4 h of head-to-toe rotation in cold room, place the tube
   on a prechilled magnet on ice and remove the supernatant.
   Do not let the beads dry out. The beads contain tagged
   ribosome–mRNA complexes. The supernatant at this step can
   be saved as the “unbound fraction.”


4. Add 500μL of wash buffer to the beads and rotate it for 5 min
   in cold room.


5. Repeat the washing step (steps 3and 4 ) at least three times
   more (total four washing steps are recommended).


6. Add 500μL of wash buffer to the beads, and transfer the buffer
   and beads to a new prechilled tube (seeNote 5).


7. Remove wash buffer from the beads using a DynaMag magnet,
   and resuspend the beads in 100μL of CHX-lysis buffer.


8. Add 350μL of RLT buffer (RNeasy mini kit) containing 2-
   Mercaptoethanol (seeNote 6). Vortex vigorously and incubate
   for 5 min at room temperature. During this step, mRNAs are
   dissociated from the tagged ribosome.


9. Centrifuge for 30 s using a benchtop centrifuge and collect the
   supernatant using a DynaMag magnet at room temperature.
   The supernatant contains axon-TRAPed mRNAs.


10. Add 250μL of 100% ethanol to the supernatant (step 9).


11. Purify RNA using RNeasy mini kit (Qiagen). Perform on-
    column DNase digestion to remove potential DNA
    contamination.


12. Elute RNA from the column in 14μL of RNase-free water (see
    Note 7).


13. Proceed to cDNA synthesis (seeNote 8).

3.5 In Vitro
Ribosome Run-Off

For the run-off experiments, we use run-off (RO)-lysis buffer that

does not contain any detergent and cycloheximide (seeNote 9).

1. Prepare the RO-lysis buffer immediately before the
   experiment.

90 Toshiaki Shigeoka et al.