title: GSM5823535: Wt_AI_tot02_SC_SP_TAATGT [2]; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Total RNA was prepared by adding acid phenol (200 ul) on the cell pellet together with 200 ul of TES (Tris/EDTA/SDS) (Current Protocols in Molecular Biology (1993)), soluble RNA was prepared by lysing the cell pellet as for polysome profiling (Panasenko and Collart, 2012, doi:10.1111/j.1365-2958.2011.07957.x), and addition of an equal volume of hot acid phenol to the lysis buffer. HT-5PSeq libraries were generated as reported (Zhang and Pelechano, doi: https://doi.org/10.1101/2020.06.22.165134) with minor modifications.   In brief, 15µg total RNA, containing 5% total RNA from Schizosaccharomyces pombe as spike-in, was used. Each sample was spited in two. One part was used for preparing conventional HT-5PSeq libraries and the other part for was random fragmented prior to the preparation of HT-5PSeq libraries (negative control).  For random fragmented samples: 7.5ug RNA was fragmentation at 80 ̊C for five minutes in fragmentation buffer (40mM Tris Acetate pH 8.1, 100mM KOAc and 30mM MgOAc). Samples were purified using 1.8x volumes of RNACleanXP beads (Beckman Coulter) following re-phosphorylation of 5'OH sites at 37 ̊C for 60 minutes with 5 Units of T4 Polynucleotide kinase (PNK, NEB). Subsequent, reaction was purified using Phenol:Chloroform: Isoamyl Alcohol (24:25:1), followed by sodium acetate-ethanol precipitation. From this step forward, experimental pipelines for random fragmented and HT-5PSeq library preparation merge.   For HT-5PSeq Libraries: 7.5 µg RNA was ligated over night at 16˚C to r5P_RNA_MPX oligo (CrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU rXrXrXrXrXrX rNrNrNrNrNrNrNrN) carrying a sample barcode (rX) and unique molecular identifiers (rN). Ligase was deactivated using 5mM EDTA and heat at 65˚C for 10 minutes (up to X individual barcoded RNA ligations were pooled) and subsequent purified using 1.8x volumes of RNAClean XP beads (Beckman Coulter). Ligated RNA was then reverse transcribed using random hexamer (5Pseq-RT, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN, 20µM) and oligo-dT (5Pseq-dT, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTTTTTT at 0.05µM) oligos to prime. After, remaining RNA was degraded using NaOH. Ribosomal RNA was removed using previously descrived rRNA DNA oligo depletion mixes, following a duplex-specific nuclease (DSN, Evrogen) digestion. rRNA depleted cDNA was amplified by PCR (17 cycles) and final product was enriched for fragments with the range of 300-500nt using Ampure XP.   Size selected HT-5P Libraries were quantified by fluorescence (Qubit, Thermo Fisher), size estimated using an Agilent Bioanalyzer and sequenced using a NextSeq500 Illumina sequencer (75cycles High output kit).  Sequencing files were demultiplexed using bcl2fastq v2.20.0.422 (one mismatch, minimum length 35nt), and adapters were trimmed using cutadapt 2.3.  at default settings, allowing one mismatch and minimum read length of 35nt. In addition to standard illumine dual index (i5, i7), the inline sample and UMI barcode was analyzed using Umitools. Reads were mapped to the concatenated genome of S. cervisiae (R64-1-1) and S. pombe (ASM294v2) using STAR.  Second read enables to splits reads between oligo-dT or random primer. That information was not used in the current analysis.