Structural basis for template switching by a group II intron-encoded reverse transcriptase

We constructed TGIRT-seq libraries with WT and F143A GsI-IIC RTs at two different dNTP concentrations (4 and 0.4 mM) in reaction medium containing 200 mM NaCl under conditions designed to exacerbate the number of secondary template switches, including the use of a very short 24-nt acceptor RNA with three randomized 3' nucleotides (denoted NNN) to enable base pairing of different nucleotides added by NTA to the 3' end of completed cDNAs.

本研究使用野生型（Wild Type, WT）与F143A突变型GsI-IIC逆转录酶（Reverse Transcriptase, RT）构建了TGIRT-seq文库，实验设置了两种不同的脱氧核苷三磷酸（dNTP）浓度（4 mM与0.4 mM），在含200 mM氯化钠的反应介质中，于旨在增加次级模板切换事件数量的条件下完成文库构建。该建库条件包含使用一段极短的24 nt受体RNA，其3'末端带有三个随机化核苷酸（标记为NNN），以允许由NTA添加至已完成合成的互补DNA（cDNA）3'端的不同核苷酸之间发生碱基配对。