Gel-free OTTR by capture of biotinylated-input OTTR cDNA duplexes. (A) The 3′-labeling activity of W403AF753A BoMoC with various d(d)RTPs resolved by 12% dPAGE and detected by the 5′ IRD800 fluorophore of the 3′NN single-stranded DNA substrate. The number in the biotinylated nucleotide names refers to the carbon linker. ddATP-11-biotin, ddGTP-11-biotin, dATP-11-biotin, and dGTP-11-biotin were biotinylated from the 7-position of the purine base while dATP-14-biotin was biotinylated from 6-position of the purine base. Red circles indicate the migration of primer without elongation. (B) Fractional content of the total sequenced reads for different miRXplore miRNA libraries with different input amounts, 3′-labeling nucleotide, and post-cDNA synthesis clean-up (i.e., either gel-based or streptavidin pull-down). (C) Scatter plot of miRXplore miRNA library CV and 3′ precision of various biotinylated OTTR libraries. The Buffer 4A (either 4A, 4A1, or 4A4) used in each library is labeled by color of the data points. Buffer 4A recipes are specified at right. The shape of the data points indicates the nucleotide and reaction condition used during 3′ labeling. Libraries with the same 3′-labeling nucleotide and buffer 4A are grouped together by a bounding line as a visual aid. Gel-based OTTR refers to OTTR v1.3 (D) Distributions of the log2 CPM of miRXplore miRNA based on miRNA 3′ nucleotide for the libraries in C, with red for purine and blue for pyrimidine, as indicated by the key. Library-wide CV for each library is given in the top left corner of the plots. Color of CV text indicates which buffer 4A variant was used. A representative gel-based cDNA purification of OTTR cDNA library is included at right.
