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.