| Full name | Ordered Two-Template Relay Sequencing |
| Summary | using retroelement RT for serial template jumping to add distinct 5′ and 3′ adaptors during cDNA library synthesis. Streamlined, automation-friendly, single-tube cDNA library production for next-generation sequencing (NGS) |
| Key findings | EVs contain an abundance of many ncRNAs that had been previously unidentified, including tRNAs and tRFs |
| Sequencing strategy | RNA-Seq |
| Raw data | https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA726257M |
| Experimental protocol | Input RNA (10 ng) from the 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 μM ddATP (±250 μM ddGTP), and 0.7 μM BoMoC and then incubated for 1.5 to 2 h at 30 °C. For ddGTP chase of initial labeling with ddATP, the reaction was allowed to proceed for 1.5 h at 30 °C with ddATP only and then chased with 250 μM ddGTP and incubated for another 30 min at 30 °C. The reaction was stopped by incubating at 65 °C for 5 min followed by addition of 5 mM MgCl2 and 0.5 units of shrimp alkaline phosphatase (NEB, M0371S). The phosphatase reaction was incubated at 37 °C for 15 min, stopped by addition of 5 mM EGTA, then incubated at 65 °C for 5 min. Subsequently, buffers were added to give an additional 0.5 mM MgCl2 and 45 mM KCl plus 2% PEG-6000, 200 μM dGTP, 40 μM dTTP and dCTP, 2 μM dATP, 150 μM DAP, 90 nM RNA-DNA primer duplex with +1T and +1C overhangs, 180 nM terminating AT, and 0.5 μM BoMoC. Samples were processed as previously discussed and then resuspended in 10 μL H2O. If Universal adaptors were used in the RT reaction, libraries were generated using 5 μL of the purified RT reaction product and 4 to 8 cycles of PCR with Q5 high fidelity polymerase (NEB, M0491S). PCR products were column purified and separated by 7.5% urea-PAGE to remove adaptor dimer, and the desired product was isolated by diffusion overnight at 37 °C followed by PCI extraction, ethanol precipitation, and resuspension in 10 μL H2O. If Full-length adaptors were used, the RT reaction was treated with RNase A and RNase H prior to cleanup, 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 to 30 nM following four cycles of PCR for the miRXplore reference standard and from 6 to 10 nM with more complex input pools (total cellular sRNA and EV RNA) following four cycles of PCR. Yield using Full-length adaptors with the miRXplore reference standard was ∼1 nM. To recover 10 M reads for total cellular and EV RNA-seq, ∼1.5% of each library was taken to produce the starting 1 nM sample pool. |
| Analysis protocol | OTTR oligo sequences were trimmed from the libraries using cutadapt and reads shorter than 10 bases were discarded. tRNA reads were determined and quantified by tRAX (59) first, following miRNAs detection and quantification by miRDeep2 (60). Validated miRNA were defined by their presence in the filtered miRGeneDB (61). The remaining reads greater than 20 bases were retained, and sequentially mapped to human rRNA (U13369.1, NR_145819.1, NR_146144.1, NR_146151.1, NR_146117.1, X12811.1, ENST00000389680.2, ENST00000387347.2, NR_003287.4, NR_023379.1, NR_003285.3, NR_003286.4), ncRNA (Ensembl), mRNA (GENCODE), lncRNA (GENCODE), and gDNA (GENCODE) using bowtie. The alignment files were merged and RSEM (62) was used to quantify read counts. Tximport and DEseq2 were used to import the counts data and estimate difference expression. Given the wide variation in library composition, analysis was largely restricted to differential expression of miRNA and tRNA. |
| Organism/cell line | extracellular vesicles from MDA-MB-231 and HEK293T |
| Approximate experimental time | 2-3 days |
| Starting RNA amount | 10 ng small RNA |
| tRNA expression | ++ |
| Base modifications | + |
| Charging status | - |
| tRNA processing and fragmentation | ++ |
| Citation | https://doi.org/10.1073/pnas.21079001 |
