title: OTTR sequencing of DRT2 RNP + 16 uM dNTP reaction: untreated
design: 10 L of 100 ng/L nucleic acids in water were heated to 95C for 2 min and immediately placed on ice. They were then mixed with 20 mM Tris-HCl pH 7.5, 200 mM KCl, 2 mM DTT, 5% PEG-8000, 2 mM ammonium sulfate, 0.7 mM sodium acetate, 2 mM MnCl2, 250 M ddATP, and 0.7 M BoMoC. After incubation at 30C for 90 min, 5 mM MgCl2 and 0.5 U of shrimp alkaline phosphatase (NEB) were added, incubated at 37C for 15 min, before addition of 5 mM EGTA and incubation at 65C for 5 min. Then, 0.5 mM MgCl2, 85 mM KCl, 200 M GTP, 40 M dTTP, 40 M dCTP, 40 M dATP, 90 nM RNA-DNA primer duplex (OTTR_c5_primer + OTTR_c5_RNA), 180 nM adapter template (OTTR_c3_AT), and 0.5 M BoMoC were added. The mixture was incubated at 37C for 20 min then 65C for 5 min. 1 L of the reaction was then directly used as template in a 50 L PCR with barcoded primers and PhusionFlash polymerase for 20 cycles and 30 seconds per elongation step. Adapter dimers were removed by pooling PCR products and purification with 0.6 volumes of SPRIselect beads (Beckman Coulter). Libraries were sequenced on a MiSeq in single-end mode (290 bp read 2).