title: GSM8238151: PolySeq~tg_15~L55~PolySeq_10min~BY4741~42C~10min~none~BR2~Poly; Saccharomyces cerevisiae; OTHER
library_name: GSM8238151
library_construction_protocol: Around 100 mg of frozen yeast that was collected by vacuum filtration was transferred to a pre-chilled 2 ml Eppendorf Safe-Lock tube. Cells were lysed with a pre-chilled 7 mM stainless steel ball (Retsch #05.368.0035) by 5x90sx30Hz pulses in a Retsch MM100 mixer mill, chilling in liquid nitrogen (LN2) between pulses. Sample was resuspended in 10:1 (v/w) polysome lysis buffer (20 mM HEPES-KOH (pH 7.4), 100 mM KCl, 5 mM MgCl2, 200 Œºg/mL heparin (Sigma #H3149), 1% triton X-100, 0.5 mM TCEP (Goldbio #TCEP25), 100 Œºg/mL cycloheximide (Sigma #C7698-5G), 20 U/ml SUPERase‚Ä¢In (Invitrogen #AM2696), 1:200 Millipore protease inhibitor IV #539136). The lysate was clarified by centrifugation at 3000 g for 30 s, and the clarified lysate was transferred to new tube and aliquots were flash frozen in LN2. A 10‚Äì50% continuous sucrose gradient in polysome gradient buffer (5 mM HEPES-KOH (pH 7.4), 140 mM KCl, 5 mM MgCl2, 100 Œºg/ml cycloheximide, 10 U/ml SUPERase‚Ä¢In, 0.5 mM TCEP) was prepared in SW 28.1 tubes (Seton #7042) using a Biocomp Gradient Master and allowed to cool to 4¬∞C. Clarified lysate (200 ¬µL) was loaded on top of the gradient, and gradients were spun in a SW28.1 rotor at 27,500 rpm for 3.5 hr at 4¬∞C. Gradients were fractionated into 0.6mL fractions using a Biocomp Piston Gradient Fractionator with UV monitoring at 254 nm, and fractions were flash frozen in LN2. UV traces were normalized to the total signal starting with the 40S peak. The samples were generated by pooling 50 ¬µL of each fraction from the free fraction (before the monosome peak) and either separately pooling the fractions with 3+ ribosomes bound and the mono/di-some fractions (for the heat shock experiments), or by combining all ribosome-bound fractions together (azide and ethanol stresses). The spike-in (50 ng of S. pombe total RNA) was then added to each pooled sample. RNA was purified via ethanol precipitation (final concentrations of 0.3 M sodium acetate pH 5.2, 0.3 ¬µg/mL glycoblue (Invitrogen #AM9516), and 70% ethanol) at -20¬∞C overnight followed by centrifugation at 4¬∞C for 30 minutes at 21,000 g. The pellet was washed with 1 mL of 70% ethanol before being resuspended in water. The purified RNA was then treated with Dnase I (NEB) before purifying again using an NEB RNA clean and concentrate kit (NEB #T2030). Sequencing libraries were prepared by from DNase I treated RNA using Qiagen FastSelect (Qiagen #334215), NEBNext Multiplex Oligos (UMI Adaptor RNA Set 1, NEB #E7335L) and NEBnext Ultra II Directional RNA library prep kits (NEB #E7760L).  Paired end 200 bp sequencing with additional reads for dual 8/8 indices plus the 11nt UMI after the i7 index was performed on an Illumina NovaSeq 6000 at the University of Chicago Genomics Facility. In general, cells were first lysed by cryo-grinding with a steel ball. When appropriate, fractionation by sedimentation or sucrose gradients was performed and RNA was dissolved in Trizol. The RNA was then purified using Zymo Direct-Zol columns. For more details, please see the protocol associated with each sample. In general, RNA sequencing libraries were prepared using either Illumina RNA sequencing kits or NEBNext Ultra II RNA sequencing kits.