GSM3204461: rad30-F35A(1-622) rnh201 rad1 rev3 rep2; Saccharomyces cerevisiae; OTHER
Genomic DNA was isolated using the MasterPure Yeast DNA Purification Kit (Epicentre) without the optional RNaseA treatment. For strains where large variation in relative ribonucleotide incorporation percentages was observed between the analyzed isolates, the analysis was repeated from cultures where the presence of the correct dNTP pool imbalance was verified  by HPLC analysis as previously described (Jia et al., 2015), and only the pool-verified samples were included in the analysis (rnh201∆; rnr1-Q288A; rnr1-Q288A rnh201∆; rnr1-Y285A; rnr1-Y285A,Q288E and rnr1-Y285A,Q288E rnh201∆). Two or more isolates were analyzed for each strain, except for rnr1-Y285A,Q288E and rnr1-Y285A,Q288E rnh201∆ where two replicates of one isolate were sequenced. RNA residues in genomic DNA from S. cerevisiae were mapped by HydEn-seq by hydrolyzing 1 µg DNA with 0.3 M KOH for 2 h at 55 °C. After ​ethanol precipitation, the DNA fragments were treated for 3 min at 85 °C, phosphorylated with 10 U of 3′-phosphatase-minus T4 polynucleotide kinase (New England BioLabs) for 30 min at 37 °C, heat inactivated for 20 min at 65 °C and purified with HighPrep PCR beads (MagBio). Phosphorylated products were treated for 3 min at 85 °C, ligated to oligo ARC140 overnight at room temperature with 10 U of T4 RNA ligase, 25% PEG 8000 and 1 mM ​CoCl3(NH3)6, and purified with HighPrep PCR beads (MagBio). Ligated products were treated for 3 min at 85 °C. The ARC76–ARC77 adaptor was annealed to the second strand for 5 min at room temperature. The second strand was synthesized with 4 U of T7 DNA polymerase (New England BioLabs) and purified with HighPrep PCR beads (MagBio). Libraries were purified, quantified with a Qubit fluorometric instrument (ThermoFisher Scientific) and 50-base paired-end sequenced on an Illumina NextSeq500 instrument, to identify the location of the free 5´-ends.
GEO Accession
GSM3204461