GSM8693245: yeast naked DNA deamination 48h replicate 2; 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 0.7 volumes of acid-washed glass beads. Beads were vortexed vigorously twice for 2-3 min each time. After adding approximately 0.1 volume 5xTE (50mM Tris-HCl, pH 7.5, 5 mM EDTA) buffer, cell lysates were centrifuged and supernatants were mixed with approximately 3 volumes of 100% ethanol and stored at -20C to allow DNA precipitiation to occur.  After centrifugation, DNA pellets were washed with 80% ethanol, dissolved in a mixture of 0.1xTE (pH7.5) and 0.17 μg/μl RNase A, and incubated at 37C for 30-40 minutes. This DNA was then quantified using a NanoDrop spectrophotometer prior to irradiation of samples. The dCPD-seq protocol was adapted from the CPDseq 2.0 method (Duan et al. PNAS 2024, 121 (4) e2310854121). Briefly, approximately 170-230mg of irradiated naked genomic wild type DNA 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 dsS2 splint adaptor labeled with biotin on the complementary 5' end. 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 dsF1 adaptors was performed. Each sample was then amplified 10 cycles in a PCR reaction with a primer set containing a unique barcode. Following amplification, libraries were cleaned and submitted to Azenta (Genewiz) for sequencing.