title: GSM4576288: mrna_wt_heat_shock(+)_rep1; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: Total RNA from 10 mL cells at 0.6 OD600 were isolated using hot acid phenol method and treated with RNase-free DNase I to get rid of residual genomic DNA contamination. Then, quantity and quality of the RNA was assessed using Qubit Fluorometer 2.0 and Bioanalyzer RNA 6000 Pico Kit, respectively. Only the RNA samples with 28S:18S ≥ 1 and the RNA integration number (RIN) ≥ 8 were used for mRNA-seq library construction. After quality checking, mRNA-seq libraries were prepared using TruSeq mRNA Sample Preparation Kit according to manufacturer's instructions. First, messenger RNAs (mRNAs) were purified from 1 μg of total RNA using oligo (dT) magnetic beads followed by fragmentation of purified mRNAs using fragmentation buffer. Second, fragmented mRNAs were subjected to first strand complementary DNA (cDNA) synthesis using random hexamer-primers, and then second strand of cDNA was synthesized using DNA polymerase I and RNase H. Third, the double-stranded cDNAs were purified using AMPure XP beads, eluted with resuspension buffer, and then subjected to 3′ end adenine nucleotide addition. Finally, sequencing adaptors were ligated to the adenylated fragments and amplified with 14 rounds of PCR. Enriched cDNA libraries were used for cluster generation and sequencing. Paired-end sequencing (75 bp x 2) were carried out for two cDNA libraries (two biological replicates of each sample) using the Illumina HiSeq 4000 sequencing platform.  To prepare Ribo-seq libraries, 200 mL yeast cells were harvested and filtered using a 0.45 μM Whatman membrane 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 grinded using Spex 6775 freezing grinding mill 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). Then, RNA concentration of the cell lysates was determined using Qubit RNA HS Assay Kit with the Qubit Fluorometer 2.0. Next, 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 for 45 min at room temperature. After that, 10 µL SUPERase*In RNase inhibitor was added to the samples to stop the digestion reaction. To pellet the ribosomes, samples were transferred to a 13 mm × 51 mm polycarbonate ultracentrifuge tube that contains 0.9 ml sucrose cushion (1 M sucrose in polysome buffer) and centrifuged at 70,000 rpm for 2 hours. Translucent ribosome pellets were resuspended in 300 µL polysome buffer followed by RNA purification using TRIzol reagent according to manufacturer's instructions. Then, samples were run on 15% polyacrylamide TBE-Urea gel for 60 min at 200 V followed by gel-purification of ~20-40 nt ribosome protected footprint (RPF) fragments using 17-34 nt length oligos as markers. Next, RPFs were treated with T4 PNK in order to heal the 2′–3′ cyclic phosphate that results from RNase I cleavage. After end-healing reaction, preadenylated adapter oligos (35 nt length) were ligated to RPFs using T4 Rnl2(tr) K227Q ligase. Then, excess adapter oligos in the reaction were depleted by adding Yeast 5′-deadenylase and RecJ exonuclease enzymes directly to the ligase reaction and incubated at 30 0C for 45 min. After that, samples were purified using Oligo Clean & Concentrator kit followed by ribosomal RNA (rRNA) depletion using yeast Ribo-Zero Gold rRNA removal kit according to manufacturer's instructions. Next, samples were reverse transcribed using Protoscript II. Then, reverse transcription products were separated from the unextended primer products by polyacrylamide TBE-Urea gel electrophoresis followed by gel-purification of the full-length reverse transcription products (80-120 nt length). Next, purified full length cDNAs were circularized using CircLigase II. Afther that, 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.