title: GSM4587146: ubi_∆ub3_∆zuo1_rep1; Saccharomyces cerevisiae; OTHER
library_construction_protocol: RNA was extracted from yeast cells using the hot phenol procedure. The amount of RNA was quantified in a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and RNA integrity was checked by agarose gel electrophoresis. HT-5Pseq libraries were prepared as in (Y. Zhang & V. Pelechano, 2020 bioRxiv) from 6 µg total RNA per sample. Briefly, the RNA was treated with TURBO DNase (Thermo Fisher) to digest DNA. After inactivation of the DNase, RNA was precipitated with ethanol and sodium acetate. The pellet was resuspended directly in ligation mix. A single-stranded RNA adapter was ligated to the RNA using T4 RNA ligase (NEB) o/n at 16 degrees C. The sample was purified with 2 volumes of RNAClean XP beads (Beckman Coulter). The purified RNA was reverse-transcribed using Superscript II (Thermo Fisher) with a combination of two custom primers, each containing a partial Illumina adapter sequence and either a random hexamer or an oligo-dT sequence. RNA was depleted with NaOH treatment at 65 degrees C. cDNA was purified using 2 volumes RNAClean XP beads (Beckman Coulter). Ribosomal cDNA was depleted using duplex-specific nuclease and specific probes. Remaining cDNA was purified using 2 volumes RNAClean XP beads (Beckman Coulter). Illumina sequencing libraries were prepared from the cDNA by PCR using Phusion high-fidelity PCR master mix (Thermo Fisher). After PCR, libraries were size-selected with 0.7 and 0.9 volumes AMPure XP beads.