Project description:Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.
Project description:We applied direct RNA long read sequencing for characterization of transcripts from constructs inserted into HEK293T mammalian cells with different promoters. Direct RNA sequencing was performed on an Oxford Nanopore GridION device using the Direct Sequencing Kit (SQK-RNA004, date accessed 15 May 2024), MinION RNA flow cell (FLO-MIN00RA), and data pre-processing was performed with MinKNOW (v24.06.10).
Project description:Using Oxford Nanopore Sequencing, we sequenced the methylome of wild type zebrafish (TL background) forebrain. Forebrains from six individuals were profiled, enabling direct detection of multiple DNA base modifications at single-base resolution. The resulting dataset includes CpG-associated 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), as well as non-CpG 5mC and N6-methyladenine (6mA). NOTE: Raw Oxford Nanopore sequencing reads have been deposited in the European Nucleotide Archive under accession PRJEB108899 - this record was created independently by the submitter, in case of access issues, please contact the submitter directly.
Project description:We performed direct cDNA sequencing in HeLa GFP∆Promoter cells by Oxford Nanopore Technology (ONT) on a MinION device to detect EGFP RNA levels after DSB induction.
Project description:We cultured MCF10a-Snail-ER cells and induced EMT initiation with tamoxifen. A matched sequencing of their PolyA RNA was performed, using Illumina and direct RNA Oxford Nanopore sequencing technologies. Both generated datasets supported the development of hybrid bioinformatics tools.