GSM8772881: W303, MS2-purified 18S: WT, replicate 2; Saccharomyces cerevisiae; OTHER
For the MS2-tagged dataset, cells were harvested by centrifugation and lysed in IXA-100 buffer (50 mM Tris-HCl pH 7.5, 100 mM KCl, 12 mM Mg(OAc)₂, 0.01% NP-40, 1 mM DTT, 100 μg/mL CHX, 1 mM PMSF, and cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche), 1 tablet/10 mL). Ribosomes carrying plasmid-derived 18S rRNA with an MS2 binding sequence were isolated by incubation with MS2 coat protein bound to anti-FLAG beads and eluted with IXA buffer containing FLAG peptide. For the cisplatin treatment dataset, cells were collected by rapid filtration through 0.45 μm membrane filters and lysed in ribo-seq lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Triton-X, 1 mM DTT, 100 µg/mL CHX, 25 U/mL TURBO™ DNase (Invitrogen)). Purified ribosomes or cell lysates (containing 10 μg of RNA) were treated with RNase I (Epicentre) at 10 U/µL at 23°C for 45 min. The reaction was quenched by placing the samples on ice and adding 10 µL of SUPERase·In™ RNase Inhibitor (Ambion). Ribosomes were sedimented through a sucrose cushion (1 M sucrose, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL CHX, 20 U/mL SUPERase·In™ RNase Inhibitor) and resuspended in 120 µL of splitting buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 1% Triton-X, 5 mM EDTA, 1 mM DTT, 100 µg/mL CHX, 20 U/mL SUPERase·In™ RNase Inhibitor). After passing through a 100K MWCO centrifugal filter unit, the flow-through fraction was collected and mixed with 300 µL of TRIzol reagent (Thermo Fisher Scientific). RNA was then purified using the Direct-zol Microprep Kit (ZYMO RESEARCH) following the manufacturer's instructions. RNA fragments of 19-34 nt were extracted from 15% TBE-Urea gels (Invitrogen) and dephosphorylated with T4 Polynucleotide Kinase (NEB). Linker oligonucleotides were pre-adenylated using the 5′ DNA Adenylation Kit (NEB) and ligated to the dephosphorylated RNA fragments using T4 RNA Ligase 2, Truncated (NEB). RNA fragments were recovered from gels and mixed with customized biotinylated oligos against specific rRNA regions. Fragments from noncoding RNA were captured with Hydrophilic Streptavidin Magnetic Beads (NEB) and discarded. The noncoding RNA-depleted RNA fragments were then subjected to reverse transcription using ProtoScript II Reverse Transcriptase (NEB), circularization with CircLigase™ II ssDNA Ligase (Epicentre), and subsequent PCR amplification using Phusion High-Fidelity DNA Polymerase (NEB) to generate libraries. Oligo Clean & Concentrator (ZYMO RESEARCH) was used throughout the library preparation to purify RNA/DNA fragments from each reaction.