Direct RNA sequencing of yeast heavy polysome (tetrasome and larger) mRNA
Yeast cells of strain BY4741 exponentially growing in YPAD media at 23C were snap-chilled by adding melting water ice followed by the addition of 2.2% w/v formaldehyde to stabilise translational complexes bound to mRNA. Following 10-minute incubation on ice with gentle mixing, the media was replaced with washing buffer by pelleting and re-suspending the cells. Cells were then pelleted and resuspended in a smaller volume of storage buffer, and the suspension droplet-frozen in liquid nitrogen. Cell wall disruption and lysis occurred by cryo-milling frozen cell suspension droplets, followed by the removal of large cell debris using low-speed centrifugation of the grindate to obtain clarified cell lysate. The separation of translational complexes contained in the clarified lysate then occurred using lysate ultracentrifugation through sucrose gradients. Upon ultracentrifugation, the ribosome, disome and trisome fractions of the polysomes were identified in the sedimentation profiles from the 254 nm absorption patterns. The identified heavy polysome (tetrasome and larger) fraction was isolated and de-proteinised, while crosslinks reversed through heating. The de-blocked RNA was then purified using SPRI beads. The purified RNA was used to construct direct RNA sequencing libraries with standard Oxford Nanopore Technologies approach based on the direct RNA sequencing kit SQK-RNA002. Oligo(dT) priming was used, and the RNA strands were sequenced using MinION Mk1B device and FLO-MIN106D R9.4.1 flowcells. Data were collected using MinKNOW version 5.5.11, basecalling was performed with Guppy version 6.5.7. Basecalled reads were aligned to the Saccharomyces_cerevisiae.R64-1-1.dna_sm.toplevel.fa using minimap2 version 2.26.