In vitro transcription and DNA-tag ligation
In vitro transcription was performed using HiScribe™ T7 Quick
High Yield RNA Synthesis Kit (NEB) with approximately 500 ng (11 pmol,
6.6 x 1012 molecules) of linear DNA library at 37 ℃
for 6 h according to the manufacturer’s protocol. The transcribed RNA
was cleaned up using the NucleoSpin® RNA Clean-up kit (Macherey-Nagel).
To anneal the mRNA library with a puromycin-FITC DNA tag, a 100 µL
reaction was set up consisting of final concentrations of 4 μM
transcribed mRNA, 6 µM puromycin-FITC DNA tag (Supplementary Table 1)
and 1 mM ATP in T4 ligation buffer (NEB). The reaction was incubated at
90 ℃ for 30 sec, and then cooled to room temperature at 1℃ per second to
correctly anneal the mRNA library with the puromycin-FITC DNA tag. Next,
3U of T4 PNK Kinase (NEB) and 20U of T4 RNA ligase (NEB) were added to
the reaction mixture and further incubated at 25 ℃ for 30 min.