Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq
Figure Lengend Snippet: Overview of template-switching experiments and determination of saturating enzyme concentrations. A , Outline of the experiments. GsI-IIC RT was pre-bound to a starter duplex (magenta) consisting of a 34-nt RNA oligonucleotide containing an Illumina Read 2 (R2) sequence annealed to a complementary 35-nt DNA primer (R2R) leaving a 1-nt, 3’-DNA overhang (N) ( Table S1 ). The 3’-over-hang nucleotide (N) base pairs with the 3’ nucleotide (N’) of an acceptor RNA (black) for template switching, leading to the synthesis of a full-length cDNA copy of the acceptor RNA with the R2R adapter linked to its 5’ end. The cDNAs were incubated with NaOH to degrade RNA and neutralized with equimolar HCl prior to further analysis. For the biochemical experiments (left branch), the R2R DNA primer in the starter duplex was 5’- 32 P-labeled (*), and the cDNA products were analyzed by electrophoresis in a denaturing polyacrylamide gel, which was dried and quantified with a phosphorimager. For RNA-seq experiments (right branch), the cDNAs were cleaned up by using a MinElute column (Qiagen; not shown) prior to ligating a 5’-adenylated R1R adapter to the 3’ end of the cDNA using the thermostable 5’ app DNA/RNA ligase (New England BioLabs). After another MinElute clean-up, Illumina RNA-seq capture sites (P5 and P7) and indexes were added by PCR, and he resulting libraries were cleaned up by using AMPure XP beads (Beckman Coulter) prior to sequencing on an Illumina NextSeq 500. B , Determination of saturating enzyme concentrations. Template-switching reactions included various concentrations of GsI-IIC RT as indicated, 50 nM RNA template/DNA primer starter duplex (5’- 32 P-labeled on DNA primer indicated by *), 100 nM of a 50-nt RNA acceptor template, and 4 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP) in reaction medium containing 200 mM NaCl at 60 °C. Aliquots were quenched at times ranging from 6 to 1,800 s, and the products were analyzed by denaturing PAGE, as described in Experimental Procedures. The numbers to the left of the gel indicate size markers (a 5’ 32 P-labeled ssDNA ladder; ss20 DNA Ladder, Simplex Sciences) run in a parallel lane, and the labels to the right of the gel indicate the products resulting from the initial template switch (1x) and subsequent end-to-end template switches from the 5’ end of one acceptor to the 3’ end of another (2x, 3x, etc .). The plot at the right shows time courses for the production of template-switching products ( i.e. , products > 2 nt larger than the primer), with each data set fit by a single-exponential function and the error bars indicating the standard deviations for three experiments. The inset table indicates the k obs and amplitude parameters obtained from the fit of an exponential function to the average values from three independent determinations, along with standard errors obtained from the fit (see Experimental Procedures).
Article Snippet: Unless specified otherwise, reactions were done with 500 nM GsI-IIC RT, 50 nM R2 RNA/R2R DNA starter duplex, and 100 nM acceptor oligonucleotide in 25 μl of medium containing 200 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl pH 7.5, 5 mM fresh DTT, and an equimolar mix of 1 mM each dATP, dCTP, dGTP, and dTTP (Promega) to give 4 mM total dNTP concentration.
Techniques: Sequencing, Incubation, Labeling, Electrophoresis, RNA Sequencing Assay, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis