In vitro transcription and ligation
After the DNA library construction, in vitro transcription was
performed, followed by column purification and ligation to the
puromycin-FITC DNA tag. Ligation was carried out via the efficient
Y-ligation method (Nishigaki, Taguchi, Kinoshita, Aita, & Husimi, 1998)
to connect the 3’ end of single strand mRNA, and the phosphorylated 5’
end of the DNA tag (Supplementary Figure 2A). Ligated products were run
on 6 % polyacrylamide TBE gel with 8 M urea to confirm the band shift
after the ligation. Incorporation of the DNA tag was confirmed by SYBR
Gold staining of the mRNA molecule and the FITC fluorescence detection
of the puromycin-FITC DNA tag (Supplementary Figure 2B). The ligation
resulted in approximately 50% of the mRNA molecules ligated to the DNA
tag. Ligated mRNA-tag product was further gel purified and electroeluted
to eliminate unligated products. The elimination of free DNA tag is
crucial for the downstream translation reaction since puromycin can
interfere with ribosome. Further mRNA-tag was recovered by ethanol
precipitation in a quantity of several µg.