Initially, the RNA or DNA of interest is purified and denoted as the input template (IT). The OTTR library preparation protocol require the IT is incubated with BoMoC, which uses terminal transferase or ‘tailing’ activity' to add a chain-terminating dideoxynucleotide base, ddRTP (ddATP or ddGTP), to the 3’ end of the IT, in the presence of manganese (Mn). To promote cDNA synthesis in the steps to follow, the divalent cation source is switched to magnesium (Mg), free ddRTPs are inactivated and dNTPs are added. Next, a RNA-DNA duplex, containing a 3’ +1Y (dTTP or dCTP) base overhang is added, allowing the RNA-DNA duplex to base pair with the ddRTP-containing IT. BoMoC extends from the 3’ of the +1Y base across the IT. Following this, dNTP concentrations are altered to encourage the addition of dGTP to the 3’ end of the cDNA IT through the non-template nucleotide addition (NTA) activity of BoMoC. A 3’ adaptor template (AT) containing a 3’ dCTP is added to the reaction, promoting base pairing between the cDNA 3’ G overhang and the 3’C base of the AT and subsequent extension by BoMoC. When using RNA as the input template, addition of RNase A and RNase H is needed to degrade remaining RNA, leaving only the cDNA template.