RNA Detection

3.2 Tissue
Preparation

In order to minimize the RNA degradation, prepare the snap-

frozen tissue samples as quickly as possible.

1. Dissect the tissues that contain the target cells or subcellular
   compartments. Dissect another tissue for genotyping (see
   Note 4). In case of RGC axons, the rostral half of the superior
   colliculi is quickly dissected.


2. Place the tissue into a prelabeled Eppendorf tube.


3. Secure the lid, and then place the tube directly into the liquid
   nitrogen.


4. The frozen tissues can be stored in a liquid nitrogen storage
   tank or 80 C freezer.


5. Genotype for the Cre transgene for each tissue sample
   prepared.

3.3 Lysis and
Preclearing

1. Prepare the CHX-lysis buffer (lysis buffer with cycloheximide)
   immediately before the use.


2. Place the frozen tissues into a Dounce homogenizer containing
   500 μL ice-cold CHX-lysis buffer (5–10 w/v % tissues in CHX-
   lysis buffer). If necessary, pull two to four tissue samples for the
   same genotype (i.e., pull Cre-positive tissues in one tube, and
   Cre-negative tissues in another tube). For RGC axons, we used
   three or four pairs of superior colliculi.


3. On ice, homogenize the tissues gently but thoroughly until
   lysate becomes clear. If using the same Dounce homogenizer
   for multiple samples, homogenize Cre-negative tissues first.
   Wash the homogenizer multiple times with RNase-free water,
   and once with CHX-lysis buffer.


4. Centrifuge the lysate at 16,000gat 4C for 10 min. Care-
   fully take the supernatant and transfer to a prechilled tube. The
   supernatant contains tagged ribosome–mRNA complexes. If
   necessary, 5μL of supernatant can be stored as the “input” for
   TRAP. During this step, prepare the magnetic Protein G beads
   as insteps 5– 7.


5. Resuspend the magnetic Protein G beads in the vial, and trans-
   fer 40μL to a new tube.


6. Place the tube on the magnet to separate the beads from the
   solution, and remove the supernatant.


7. Resuspend the beads in 40μL of CHX-lysis buffer and repeat
   thestep 6.


8. Transfer the supernatant after centrifugation (step 4) to the
   washed beads (step 7).


9. Rotate the tube (head-to-toe rotation) for 1 h in cold room
   (preclearing step to minimize nonspecific binding of mRNAs
   to the Protein G beads).

Isolation of In Vivo Axonal Translatome 89