Direct long-read RNA sequencing of slow- and fast-sedimenting yeast RNA fractions in normal growth and under glucose starvation stress

Translational control is important in all life but remains a challenge to accurately quantify. When ribosomes translate messenger (m)RNA into proteins, they attach to the mRNA in series, forming poly(ribo)somes (polysomes). It may be difficult to accurately characterize polysomes using short-read sequencing methods, due to the ambiguity of mapping and uncertainty of mRNA integrity effects. Furthermore, DNA amplification and cDNA-based techniques are prone to sequence-specific biases, and do not provide a handle towards the original primary structure of mRNA. To address these limitations in a model translation system of normally-growing, as well as glucose-starved yeast cells, we used a combination of rapid cell fixation, ribosome-associated RNA extraction, separation of ribosome-associated RNA into slow- and fast-sedimenting fractions, RNA cross-link deblocking and nanopore direct RNA sequencing (DRS). Briefly, independent duplicates of wild-type yeast Saccharomyces cerevisiae cells were grown in rich glucose-supplemented media, and then split into two sub-samples. The non-starved sub-sample has been snap-chilled and fixed with formaldehyde directly in the media, similarly to how it is performed in Translation Complex Profile Sequencing (TCP-seq) method. The starved sub-sample has been first media-replaced with the media containing no added glucose, incubated for 10 minutes more under the otherwise normal growth conditions, and then fixed as the other half. Fixed cells have then been cryo-lysed and lysate separated using ultracentrifugation through sucrose gradients. Fractions corresponding to the slow-sedimenting ribosome, disome, trisome region (RDT), and fast-sedimenting tetrasomes and higher order polysomes (PS) were then isolated. RDT and PS complexes were crosslink-deblocked and their nucleic acids purified. DRS libraries were then constructed using the RDT and PS nucleic acid material, and subjected to nanopore sequencing. DRS reads were also base-called and mapped to the reference Saccharomyces cerevisiae genome, using contemporary tools and data. Here, we provide the original raw DRS reads as FAST5 files, as well as the basecalled FASTQ and mapped BAM files, for each replicate and condition. These data can be used to investigate translational involvement of yeast mRNAs in normality and under the conditions of glucose stress, as well as assess the primary structure of mRNA in detail, including identification of any modified nucleotide residues. The provided data contribute to the characterization of eukaryotic translatome and epitranscriptome.