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.