Miltenyi Biotec miRXplore miRNA reference standard, PCR-free OTTRFL
Input RNA (10 ng) (miRXplore Universal Reference Standard [Miltenyi Biotech, 130-094-407], EV sRNA, or mirVana size-selected total cellular RNA) was diluted into 20 mM Tris-HCl pH 7.5, 150 mM KCl, 0.5 mM DTT, 5% PEG-8000, 2 mM MnCl2, 250 M ddATP (+/- 250 M ddGTP), and 0.7 M BoMoC then incubated for 1.5-2 h at 30C. For ddGTP chase of initial labeling with ddATP, the reaction was allowed to proceed for 1.5 h at 30C with ddATP only, then chased with 250 M ddGTP and incubated for another 30 min at 30C. The reaction was stopped by incubating at 65C for 5 min followed by addition of 5 mM MgCl2 and 0.5 units of Shrimp Alkaline Phosphatase (rSAP, NEB M0371S). The phosphatase reaction was incubated at 37C for 15 min, stopped by addition of 5 mM EGTA, then incubated at 65C for 5 min. Subsequently, buffers were added to give an additional 0.5 mM MgCl2 and 45 mM KCl plus 2% PEG-6000, 200 M dGTP, 40 M dTTP and dCTP, 2 M dATP +/- 150 M 2-amino-2-deoxyadenosine-5-triphosphate (DAP), 90 nM RNA-DNA primer- duplex with +1T and +1G overhangs, 180 nM terminating AT, and 0.5 M BoMoC. Samples were processed as above then resuspended in 10 l H2O. If Universal adaptors were used in the RT reaction, libraries were generated using 5 l of the purified RT reaction product and 4-8 cycles of PCR with Q5 high fidelity polymerase (NEB, M0491S). PCR reaction products were column purified, separated by 7.5% urea-PAGE to remove adaptor dimer, and the desired product was isolated by diffusion overnight at 37C followed by PCI extraction, ethanol precipitation, and resuspension in 10 l H2O. If Full-length adaptors were used, the RT reaction was treated with RNaseA and RNaseH prior to clean-up and no PCR amplification was performed. Quantification of libraries prior to sequencing used qPCR with primers specific to the Illumina P5 and P7 adaptor sequences and standards from the NEBNext Library Quant Kit (NEB, E7630S). Sequencing of prepared libraries was performed using an Illumina MiniSeq with the 75-cycle high-output kit. Library yield using Universal adaptors ranged from 20-30 nM following 4 cycles of PCR the miRXplore reference standard and from 6-10 nM with more complex input pools (total cellular sRNA and EV RNA) following 4 cycles of PCR. Yield using Full-length adaptors with the miRXplore reference standard was ~ 1 nM.