GSM1521155: Pol3LG,rnh201.2; Saccharomyces cerevisiae; OTHER
DNA was isolated using the MasterPure™ Yeast DNA Purification Kit (Epicentre) without RNase A treatment. Ribo-seq was performed by hydrolyzing one μg of genomic DNA with 0.3 M KOH for 2 hours at 55°C. Following ethanol-precipitation, the DNA fragments were treated for three minutes at 85°C, phosphorylated with 10 units of 3´-phosphatase-minus T4 polynucleotide kinase (New England Biolabs) for 30 minutes at 37°C, heat inactivated for 20 minutes at 65°C and purified using HighPrep™ PCR beads (MagBio). Phosphorylated products were treated for three minutes at 85°C, ligated to oligo ARC140 (Supplementary Table 1) overnight at room temperature using 10 units of T4 RNA ligase, 25% PEG8000 and 1 mM CoCl3(NH3)6, and purified using HighPrep™ PCR beads (MagBio) . Ligated products were treated for 3 minutes at 85°C. The second strand was synthesized using four units of T7 DNA polymerase (New England Biolabs). The ARC76/ARC77 adapter was annealed to the second strand for five minutes at room temperature and purified using HighPrep™ PCR beads (MagBio). Libraries were PCR amplified using primer ARC49 and primer ARC79 or ARC84 to ARC107, using KAPA HiFi Hotstart ReadyMix (KAPA Biosystems). Libraries were then purified using HighPrep™ PCR beads (MagBio) and pooled for sequence analysis. Paired-end sequencing was performed on a HiSeq2500 sequencer (Illumina) or MiSeq to identify the location of the 5´-DNA ends generated by alkaline hydrolysis.