Reverse transcription for cDNA synthesis
Reverse transcription (RT) was performed against purified mRNA-peptide conjugate to obtain mRNA/cDNA duplexes for cDNA display using PrimeScript One Step RT-PCR Kit Ver.2 (Takara Bio). The initial peptide library (0th round) was prepared by mixing FLAG-random and 10aa-random mRNA-peptide conjugate samples to a 1:10,000 molar ratio. Approximately 200 ng (~2 x 1012 molecules) of the mRNA-peptide initial library was reverse transcribed in 50 µl reaction volume with final 0.4 µM of RTPCR-R primer in 1 step buffer (Takara Bio) supplied by the kit. Tubes were incubated at 80 ℃ for 30 seconds, then cooled down to 4℃ for primer annealing. After, the PrimeScript 1 step Enzyme Mix (Takara Bio) was added and incubated at 48 ℃ for 20 min. We also prepared RT(–) tube as a negative control without adding the enzyme mix. After the incubation, both samples were purified using the NucleoSpin RNA Clean-up XS kit (Macherey-Nagel) and quantified by Nanodrop.