GSM9523122: zuo1 S288C, MG132 + AZC 45min, biol rep 1, flowcell 1; Saccharomyces cerevisiae; OTHER
Cell pellets were resuspended in 200 µL Phenol/LET (100 mM LiCl, 20 mM EDTA, 25 mM Tris-HCl pH 8) and approximately 200 µL of glass microbeads were added to lyse the cells. After vortexing for 5 minutes at room temperature, 300 µL of Phenol/Chloroform/LET and 300 µL DEPC-treated water was added. After 5 minutes of additional vortexing, and spinning down for 5 minutes at 16,000 x g at room temperature, aqueous phase was collected and added to 500 µL Phenol/Chloroform/LET, vortexed for 5 minutes and spun down. Aqueous phase was collected and added to 500 µL Chloroform, vortexed for 5 minutes, and spun down. Aqueous phase was collected, 40 µL of 3 M Sodium Acetate (final concentration 0.3 M), 1 mL 100% ethanol was added to precipitate RNA at -20°C for approximately 1 hour. RNA was pelleted by spinning at 16,000 x g for 20 minutes at room temperature, washed with 75% ethanol, air dried, and resuspended in DEPC-treated water. or MG132 and AZC treatment, both wild-type and zuo1Δ strains carried a PDR5 deletion to enable MG132 treatment. Cells were grown to an OD600 of 0.5 to 0.7 in 100 mL YPD media, and two 50 mL aliquots of culture were each treated with 100 µM MG132 with or without 2 mg/mL AZC at 24°C for 45 minutes. Upon collection, cultures were filtered using a 0.45 µm Whatman membrane (GE Life Sciences, USA) using a vacuum filter apparatus. After filtration, Cells were immediately scraped from the filter with a straight-edge metal spatula and plunged into 50 mL liquid nitrogen. Next, samples were ground using a Spex 6775 freezing grinding mill (Spex sample prep, USA) in the presence of 10 mL lysis buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL cycloheximide, 1% Triton X-100, 25 U/mL DNase I). The RNA concentration of each cell lysate was determined using Qubit RNA HS Assay Kit (ThermoFisher Q32852) with the Qubit Fluorometer 2.0 (Invitrogen, USA). For the ribosome profiling experiments under heat and oxidative stress, 30 µg total RNA was diluted to 200 µL with polysome buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL cycloheximide) and digested using 15 U RNase I (EpiCentre, USA N6901K) for 45 minutes at room temperature. Then, 200 U SUPERase*In RNase inhibitor (Invitrogen, USA AM2694) was added to quench the digestion reaction. To pellet the ribosomes, samples were transferred to a 13 mm × 51 mm polycarbonate ultracentrifuge tube containing 0.9 mL sucrose cushion (1 M sucrose in polysome buffer) and centrifuged at 70,000 rpm for 2 hours. For the ribosome profiling experiments under AZC treatment, 73.2 µg total RNA was diluted to 300 µL with polysome buffer and digested using 2.93 U RNase I (EpiCentre, USA N6901K) for 10 minutes at room temperature. Then, 39.2 U SUPERase*In RNase inhibitor (Invitrogen, USA AM2694) was added to quench the digestion reaction. To pellet the ribosomes, samples were transferred to a 13 mm × 51 mm polycarbonate ultracentrifuge tube containing 0.7 mL sucrose cushion (1 M sucrose in polysome buffer) and centrifuged in Beckman TLA-120.2 rotor at 55,000 rpm for 1.5 hours. Translucent ribosome pellets were resuspended in 300 µL polysome buffer followed by RNA purification using TRIzol reagent (Invitrogen, USA 15596026), according to the manufacturer's instructions. Samples were size-separated by running on 15% polyacrylamide TBE-Urea gels for 60 minutes at 200 V followed by gel-purification of the approximately 20 - 40 nucleotide (nt) length ribosome protected fragments (RPFs) using 17 - 34 nt length oligonucleotides as size markers. RPFs were treated with T4 PNK (NEB M0201S) to heal the 2′-3′ cyclic phosphate that results from RNase I cleavage. After end-healing reaction, pre-adenylated adapter oligonucleotides (35 nt length) were ligated to RPFs using T4 Rnl2(tr) K227Q ligase (NEB M0351S). Excess adapter oligos in the reaction were depleted by adding yeast 5′-deadenylase (EpiCentre, USA DA11101K) and RecJ exonuclease (EpiCentre, USA RJ411250) enzymes directly to the ligase reaction and incubated at 30°C for 45 minutes. Samples were purified using Oligo Clean & Concentrator kit (Zymo Research D4060) followed by ribosomal RNA (rRNA) depletion using either yeast Ribo-Zero Gold rRNA removal kit (Illumina, MRZG12324) for heat and oxidative stress samples or QIAseq FastSelect rRNA Yeast kit (Qiagen 334215) for AZC treated samples according to the manufacturer's instructions. Ligated RNA samples were reverse transcribed using Protoscript II RT (NEB M0368). Reverse transcription products were separated from the unextended primer products by polyacrylamide TBE-Urea gel electrophoresis followed by gel purification of the full-length cDNAs (80-120 nt length). Size-selected cDNAs were circularized using CircLigase II (EpiCentre, USA CL9021K). 10-15 rounds of PCR enrichment of the libraries were performed with the proper forward and reverse primers. Enriched libraries were used for cluster generation and sequencing. Single-end (50 bp) sequencing was carried out for two cDNA libraries (two biological replicates of each sample) using the Illumina HiSeq 4000 sequencing platform (Illumina, USA).