Ribo zero gold rrna removal kit yeast

For RPF analyses, preparation of cell extracts, RNase treatment, separation of samples by centrifugation through sucrose gradients, and isolation of protected RNA fragments were performed as previously described^{21 (link)}. Gel purified RNA fragments were treated with 10 units of T4 PNK (Thermo Fisher) in a low pH buffer (700 mM Tris pH 7, 50 mM DTT, 100 mM MgCl₂) for 30 min at 37 °C. ATP and buffer A (Thermo Fisher) were then added for an additional 30 min incubation. RNA fragments were column-purified (PureLink RNA micro-columns, Life Technologies). 100 ng were used as input for the NEXTflex Small RNA Sequencing Kit v2 (Bioo Scientific), and libraries were generated following the manufacturer’s protocol. For mRNA analyses, total RNA was isolated as previously described^{21 (link)}. rRNA was then removed from total RNA using Ribo-Zero Gold rRNA Removal Kit Yeast (Illumina), with 4 µg as input. Finally, 30 ng of rRNA-depleted RNA was used as starting material for the NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific).

Single colony isolates of each strain were grown to midlog phase in 50 ml of liquid YPD media. Samples were then pelleted and washed once with sterile water before being flash frozen in liquid nitrogen and stored for 16 hr at −80°. Samples were thawed on ice, and RNA was extracted using a phenol freeze-based approach as previously described (Schmitt et al. 1990 (link)). The extracted RNA was subsequently treated with RNase-free DNase I (Thermo Fisher Scientific).
RNA samples were processed and sequenced at the BC Cancer Agency Michael Smith Genome Sciences Centre following standard operating protocols. Briefly, total RNA samples were ribo-depleted using the Ribo-Zero Gold rRNA Removal Kit (Yeast) (Illumina, San Diego, CA) and analyzed on an Agilent 2100 Bioanalyzer using Agilent 6000 RNA Nano Kit (Agilent Technologies, Santa Clara, CA). Complementary DNA (cDNA) was generated using the Superscript Double-Stranded cDNA Synthesis kit (Thermo Fisher) and 100 bp paired-end libraries were prepared using the Paired-End Sample Prep Kit (Illumina).