Multiplexed nanopore direct RNA sequencing of tRNAs from four species using Nano-tRNA-seq and WarpDemuX-barcoding

This study presents nanopore direct RNA sequencing data from multiplexed tRNA populations of four diverse species: Homo sapiens (HEK293 cells), Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans. The experiment utilized a novel multiplexing approach called WarpDemuX-tRNA, which incorporates unique DNA barcodes into the RTA adapter during library preparation. Total RNA was extracted from each species, followed by small RNA isolation and tRNA deacylation. Libraries were prepared using the Nano-tRNA-seq protocol combined with WarpDemuX barcoding, where equal amounts (200-500 ng) of deacylated tRNAs were ligated to splint adapters and barcoded RTA adapters. The samples were reverse transcribed using Induoro reverse transcriptase and sequenced on Oxford Nanopore Technologies P24 A-series devices with FLO-PRO004RA flow cells. Samples were independently sequenced, with different species-to-barcode mappings between replicated. Sequencing was conducted using live basecalling with Dorado Basecall Server 7.4.13 (MinKNOW 24.06.14) using the High Accuracy model with a Q-score threshold of 7. We compiled a database of reference sequences from gtrnadb.ucsc.edu. All tRNA reads were aligned to this reference using Parasail (version 2.6.0). We filtered the resulting alignments using a 99.9% specificity threshold.