title: GSM8944817: YS602_mH69_5Pseq_rep2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Cells were lysed using glass beads and total mRNA was extracted using phenol–chloroform. 50 ug of total mRNA was used as the starting material to perform 5PSeq. Poly(A)-containing mRNA was enriched using 100 µl oligo(dT) beads (Dynabeads oligo (dT)25, Life Technologies) for 5P samples while randomly generated fragments of total RNA were prepared as controls. Both 5P and control RNAs were fragmented at 80˚C for 5 min. Following reverse transcription and second strand generation, overnight incubation at 4 °C with M-280 streptavidin beads (Life Technologies) was performed instead of 30 min at room temperature, as suggested for maximum capture of the cDNA. The final DNA adapter ligation reaction was carried out for 2hrs at 16˚C. Following PCR, each sample was purified separately with HighPrep beads (Magbio) as suggested and their quality was checked using Agilent 2100 Bioanalyzer. A Qubit 2.0 Fluorometer (Life Technologies) was used to determine the concentration of each library. Libraries were pooled in equal molar ratios and fragments were enriched between 300 and 500 nt for the library. The final libraries were sequenced using Illumina NovaSeq 6000.