Extension lengths of cDNAs using double-stranded input material to DragonRNA Rpo41 reactions. Rpo41 reactions were run for 30 min under “NEPol conditions” with double-stranded DNA inputs containing a template oligonucleotide and a primer oligonucleotide. The primer oligonucleotide could not self-prime. Sequencing was performed using DruSeq (see “Materials and methods” section). Plots show distribution of extension lengths on the DNA primer from the sequencing results. (A) Cartoon showing design of input oligonucleotides with expected annealing. (B and C) Proposed model for DragonRNA extension on double-stranded input involves RNAP helicase activity. Proposed model for RNAP displacing the second strand on a hairpin-loop double-stranded, annealed DNA input in a DragonRNA reaction to extend the DNA primer oligonucleotide with RNA. This is shown in panel (B) with a hairpin-loop double-stranded region in a system containing a gap of 3 bases and in panel (C) with a three-oligonucleotide double-stranded input with a nick and no gap. (D) Extension lengths from sequencing of reaction with template DNA oligonucleotide that was a single molecule with a hairpin loop region and a 3-base gap between the 5′ end of the template molecule and the 3′ end of the primer molecule. (E) Extension lengths from sequencing of reaction with template DNA oligonucleotide that was a single molecule with a hairpin loop region and a 0-base nick between the 5′ end of the template molecule and the 3′ end of the primer molecule. (F) Extension lengths from sequencing of reaction with template DNA oligonucleotide that was a single molecule template, an annealed DNA oligonucleotide to provide a double-stranded region, and a 3-base gap between the 5′ end of the double-stranded region and the 3′ end of the primer molecule. (G) Extension lengths from sequencing of reaction with template DNA oligonucleotide that was a single molecule template, an annealed DNA oligonucleotide to provide a double-stranded region, and a 0-base nick between the 5′ end of the template molecule and the 3′ end of the primer molecule. (H and I) RNA-sequencing example reads for Rpo41 30-min reactions with double-stranded inputs, one made from hairpin looped DNA (H) and one made from a three-oligonucleotide system (I). The sequences show examples of reads that extend part-way complementary to the template strand sequence. Figure shows the two exemplary sequence species after filtering for 5′ matches to the DNA primer sequence with putative base pairing and templating configurations drawn for purposes of illustration. Top text in the graphic indicates the primer DNA sequence. Bottom text in the graphic indicates the template sequence or nonprimer oligonucleotides. The two bases on the 3′ end of the DNA input oligonucleotide primer are indicated with color. We expect that sequences downstream from the purple text are produced in the polymerase reaction from rNTP precursors. Text without base pairing indicates putative RNA extension on the DNA primer. Note the library preparation involved converting the RNA to cDNA, and thus the sequencing reads shown here (even regions presumed to be RNA) are shown with “T.” Gel assay showing extension activity for this sequencing is shown in Supplementary Fig. S10.