GSM9348038: Total RNA, biol rep3, glucose replete condition, timepoint 5; Saccharomyces cerevisiae; OTHER
Total RNA extraction: Cell pellets were snap-frozen and lysed in TRIzol with glass bead disruption, followed by phenol-chloroform extraction, isopropanol precipitation, DNase I treatment, and ethanol purification. RNA quality was assessed by Qubit (RNA HS) and TapeStation 4200.  P-body RNA extraction: Cells were lysed in Blob100 buffer with RNase inhibitors and protease inhibitors. High-molecular-weight complexes were pelleted, and P-bodies were immunoprecipitated using anti-Myc magnetic beads. After stringent Blob500 and Blob100 washes, bound RNA was released by Proteinase K treatment, crosslinks reversed, and RNA extracted using the RiboPure Yeast Kit. DNase I treatment and purification were performed as above. Incorporated 4TU residues were alkylated with 10 mM iodoacetamide in 30% DMSO at room temperature for 15 min, quenched with DTT, and ethanol-precipitated. Strand-specific 3′ mRNA libraries were prepared using the Lexogen QuantSeq 3′ mRNA-Seq V2 (FWD) kit with Unique Dual Indices. Libraries were amplified (13–15 PCR cycles), purified, quantified, and sized prior to pooling. Sequencing was performed on an Illumina NovaSeq 6000.