GSM4849020: Msp1_Pex19_RNA; Saccharomyces cerevisiae; RNA-Seq
All cultures were rapidly harvested by vacuum filtration and flash frozen in liquid nitrogen as described in Weinberg et al., 2016 (GSE53268). Frozen pellets were mechanically lysed using a Spex SamplePrep Freezer/Mill (10 cycles of 2 min on, 2 min off at setting 10). Lysate powder was aliquoted and stored at –80°C. For RNA-seq samples, total RNA was extracted from lysate powder using TRI Reagent (Ambion) according to the manufacturer's protocol. rRNA was depleted from 5 μg of total RNA using the Ribo-Zero Gold Yeast rRNA Removal Kit (Illumina) according to the manufacturer's protocol. For RPF samples, lysate powder was resuspended in a buffer containing cycloheximide and tigecycline in order to arrest translating ribosomes. Monosomes were isolated by treating the lysate with RNase I followed by purification over a 10-50% sucrose gradient. Ribosome-protected fragments were further isolated by Proteinase K/SDS digestion and phenol/chloroform extraction.     Two quantitative spike oligos were added to each library to access gross differences in absolute levels of ribosome-protected fragments between samples (Spike_21: GAUGUAACGAGUUGGAAUGCA/3pp/, Spike_28: CAUCGGUCGAACUUAUGUGAAACAUCAU/3pp/, 2 fmol each spike per sample). RNA-seq samples were fragmented by alkaline hydrolysis and size-selected in the range of 19-40 nt. RPF samples were size-selected in the range of 19-33 nt, and contaminating rRNA sequences were removed via subtractive hybridization. RNA-seq and RPF samples were both ligated to adaptors, reverse-transcribed, and amplified to prepare sequencing libraries as described in Subtelny et al. 2014 (GSE52809). Libraries were sequenced on an Illumina HiSeq 2500 using 60-bp single-end reads.