GSM8862816: hap5_UV_0hr_cellular_replicate1; Saccharomyces cerevisiae; OTHER
Yeast genomic DNA was extracted by mixing cell pellets with approximately equal volumes of DNA lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 7.5-8.0, 1 mM EDTA) and PCI (Phenol:Chloroform:Isoamyl Alcohol=25:24:1), and approximately 0.5 volumes of acid-washed glass beads. Beads were vortexed vigorously for about 4 min. After adding TE (10mM Tris-HCl, pH 7.5-8.0, 1 mM EDTA) buffer, cell lysates were centrifuged and the supernatant was mixed with 2.5-3 volumes of 100% ethanol and stored at -20C to precipitate the DNA.  DNA pellets were washed with 75-80% ethanol, dissolved in mixture of 1xTE (pH7.5) and 0.2 μg/μl RNase A, and incubated at 37C for approximately 1 hr. The purified genomic DNA was then used for each sequencing library preparation. The CPD-seq method was employed as previously described (Mao et al., PNAS 2016, 113 (32) 9057-9062). Briefly, approximately 50ug of yeast genomic DNA (gDNA; as measured by NanoDrop spectrophotometry) was extracted from UV-exposed cells via the phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation method. Genomic DNA was then sonicated (30s ON/OFF; 25 cycles; Diagenode Biorupter 300) to generate ~200-600bp fragments and ethanol precipitated. Fragmented DNA was converted to blunt-ended DNA with 5' phosphates and 3' hydroxyls with the NEBNext End Repair Module (NEB) and dA-tailed for ligation with the NEBNext dA-Tailing Module (NEB). A first double-stranded i7 Illumina adapter was ligated onto blunted 3' hydroxyls. Any remaining free 3' hydroxyls were blocked via incubation with terminal transferase (NEB) and either dideoxy ATP or GTP.  T4 endonuclease V (NEB) and APE1 were used to cleave at sites of CPDs and generate new ligatable 3' hydroxyl ends. A second double-stranded, biotinylated i7 Illumina adapter was ligated to these free 3' hydroxyls. Streptavidin beads were used to capture fragments containing the biotinylated second adapter, a wash with 1X SSC (saline sodium citrate) buffer removed non-specific binding, and 0.15M NaOH was used to elute the DNA prior to ethanol precipitation. The complementary strand was synthesized with EconoTaq (Lucigen) and the resulting libraries were amplified with 5 cycles of PCR containing unique barcoded primers for each sample. Libraries were pooled and submitted to Azenta for either Illumina HiSeq 4000 or Illumina NovaSeq X Plus sequencing. Cytiva SeraMag Select beads were used to purify DNA between enzyme incubation steps.