TUCL-Seq of Homo sapiens: synthetic miRNA mixture, 0.1 pmol, replicate 2
Initially, a poly(A) tail is added to 3' end of the miRNAs using PAPase, followed by direct capture via poly(dT50LNA2) probes on the SMS flow cell. On the flow cell, reverse transcriptase converts RNA to cDNAs; Exo I digests excess probes. After denaturation and miRNA elution, TdT adds 2~4 rU to the 3' end of 1st cDNA, proteinase K removes adsorbed TdT, and T4 RNA ligase 1 links the ssDNA sequencing adapter to the 1st cDNA 3' end. Sequencing primer hybridizes, polymerase fills single-stranded regions, and sequencing proceeds.