GSM8677935: rad14_cdc13-1_cellular_0hr_rep1; 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 twice for about 2 min each time. 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 allow DNA precipitiation to occur.  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 15 minutes. The purified genomic DNA was then used for each sequencing library preparation. The dCPD-seq protocol was adapted from the CPDseq 2.0 method (Duan et al. PNAS 2024, 121 (4) e2310854121). Briefly, approximately 250mg of yeast gDNA (measured by NanoDrop spectrophotometry) was incubated with TdT (NEB) and either ddATP or ddGTP to block free 3' OH ends. After phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation, the DNA was resuspended in water and 10x CPD photolyase buffer (100mM NaCl, 50mM Tris HCl pH 7.5, 20mM DTT, 1mM EDTA, 50% glycerol) to a final concentration of ~1x buffer, and E. coli CPD photolyase and UDG were added to reverse CPDs and excise uracil bases. Reactions were incubated under 365nm light (Spectroline EA-160) to reverse CPDs and then phenol:chloroform:isoamyl alcohol extracted and ethanol precipitated. APE1 (NEB) was then used to cleave the abasic sugar backbone to yield free 3'OH ends that were ligated to a biotinylated splint adaptor that labeled with biotin on the complementary 5' end. Up to six different barcoded A adaptors were used to generate the library.  Ligated DNA was captured with streptavidin beads (Invitrogen), washed with 1x SCC (saline sodium citrate) buffer to remove nonspecific binding, and the non-biotinylated strand containing the ligated 3' A adaptors was eluted with 0.15M NaOH. After synthesis of the complementary strand with EconoTaq (Lucigen) and preparation of DNA ends with NEBNext end repair and NEBNExt dA-tailing modules, a second adaptor ligation with trP1 adaptors was performed. The library was then amplified 7 cycles in a PCR reaction with primers complimentary to the A and trP1 adaptors. Libraries were submitted to the WSU LBB1 Core Facility for Ion Torrent sequencing.