RNA Detection

4. If genotyping is needed, prepare tail or toe biopsies for geno-
   mic DNA PCR experiments. Clean the equipment to avoid
   carry-over/cross contamination between samples.


5. This step can reduce the false-positive signals due to nonspe-
   cific binding of mRNAs to the tube.


6. Alternatively, you can carry out a competitive elution using
   excessive HA peptides (Sigma, I2149). Afterstep 7of Sub-
   heading3.4, resuspend the beads containing the tagged ribo-
   some–mRNA complexes in 100μL of 100μg/mL HA peptide
   in CHX-lysis buffer. Vortex vigorously and incubate for 5 min
   at room temperature. Take the supernatant on a DynaMag
   magnet and transfer the supernatant to a prechilled tube on
   ice. The supernatant contains axon-TRAPed mRNAs. Proceed
   tostep 8. Omitstep 9(as the bead is removed). Although the
   competitive elution reduces the total amount of purified
   mRNAs, it can improve the signal-to-noise ratio by increasing
   the specificity.


7. We estimated that approximately 40% of HA-tagged translating
   ribosomes could be purified using this protocol.


8. If abundant mRNAs are isolated, they can be sequenced by
   typical RNA sequencing protocols. Alternatively, TRAPed
   mRNAs can be amplified by a PCR-based method before
   sequencing. We amplified cDNA by the method developed by
   Azim Surani and colleagues for single cell transcriptomics,
   which utilizes PCR-based amplification of polyadenylated
   RNAs [11]. We followed the detailed protocol up to Step 41
   described in this paper [11] to make double-strand DNAs for
   deep sequencing. Successful axon-TRAP can be validated by
   agarose gel electrophoresis of cDNAs amplified from Cre-
   positive and Cre-negative samples. Cre-positive samples should
   generate stronger bands (Fig.3).

Retina SC

Unrecombined

Recombined

(Rpl22-HA)

Cre

e1 e2 e3 e4

Cre

RiboTag

e4-HA

e1 e2 e3

Cre (expressed in retina)

e4-HA

RiboTag fwd >>> <<< HA rev

Fig. 2PCR-based detection of Cre-mediated recombination. Cre-mediated recombination converts the normal
RiboTag allele to the HA-tagged, recombined allele (seethe gene model on theright). These two alleles can be
distinguished by the primers depicted in the diagram. Genomic DNAs were extracted from the retina
(containing the cell bodies of RGCs) and the superior colliculus (SC, containing the axon terminals of RGCs)
of RiboTag mice with or without Pax6-alpha-Cre allele. Cre-mediated “RiboTag” is detectable only in the
retina but not in the superior colliculus

92 Toshiaki Shigeoka et al.