Yeast Hydrolysis-Enabled Sequencing (HydEn-seq)

The HydEn-seq protocol was performed essentially as previously described (3 (link)). Briefly, mid-log phase yeast cells from independent cultures were harvested and genomic DNA (gDNA) isolated using the MasterPure Yeast Purification Kit (Epicentre, no RNase A treatment). 1 μg of gDNA was hydrolyzed with 0.3 M KOH for 2 h at 55°C. DNA fragments were purified using ethanol precipitation, denatured and phosphorylated with 10 U of 3′-phosphatase-minus T4 polynucleotide kinase (New England BioLabs) for 30 min at 37°C, followed by heat inactivation at 65°C for 20 min. DNA was purified using CleanPCR beads (CleanNA), denatured and ligated to oligo ARC140 overnight at room temperature with 10 U T4 RNA ligase (New England BioLabs), 25% PEG 8000 and 1 mM CoCl₃(NH₃)₆. After DNA purification with CleanPCR beads and denaturing, the ARC76-ARC77 adaptor was annealed for 5 min at room temperature. Second-strand synthesis was performed using 4 U of T7 DNA polymerase (New England BioLabs) and DNA purified with CleanPCR beads. Libraries were PCR amplified with primer ARC49 and index primers ARC79 to ARC107 using the KAPA HiFi Hotstart ReadyMix (KAPA Biosystems). Libraries were purified with CleanPCR beads and pooled for sequencing. 75-bp paired-end sequencing was performed on an Illumina NextSeq500 instrument, to locate 5′-ends generated by alkaline hydrolysis.