title: GSM6601607: rep2_naked_WT_yeast_naked_500JUVC_UVDE-seq_UGI_A6_1bp; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Yeast genomic DNA was extracted by mixing cell pellets with 250 μl of DNA lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA), 250 μl of PCI (Phenol:Chloroform:Isoamyl Alcohol=25:24:1), and 150 μl of acid-washed glass beads, and vortexed vigorously twice, 2 min each time. After adding 200 μl of TE (10mM Tris-HCl, pH 7.5, 1 mM EDTA) buffer, cell lysates were centrifuged and the supernatant was mixed with 1 ml of ethanol to precipitate DNA at room temperature for 5 min. DNA pellet was dissolved in 200 μl of TE and incubated with 2 μl of RNase A (ThermoFisher Scientific) at 37˚C for ~1 hr to remove RNA. DNA was subsequently purified with PCI extraction, ethanol precipitated, and dissolved in sterile deionized H2O. UVDE-seq procedure is adopted from the published emRiboSeq method (Nature Protocols 2015 10, 1433–1444) and our own CPD-seq method. Briefly, DNA fragments were first ligated to a double-stranded adaptor DNA trP1, and free 3'-OHs were blocked by  terminal transferase and dideoxy-ATP or dideoxy-GTP.  CPD lesions were repaired using purified E. coli CPD photolyase. All other UV-induced lesions were cleaved with purified T. thermophilus UVDE. DNA was then ligated to adaptor  A. Up to 6 different barcoded A adaptors (A1: AAGAGGAT; A2: TTCGTGAT; A3: CCTGAGAT; A4: ATCGCGAT; A5: TACTGGAT; and A6: GAACTGAT) were used to generate libraries for different experiments. DNA was purified with Streptavidin beads and amplified for up to 8 cycles in a PCR reaction with primers complimentary to A and trP1, and the resulting DNA libraries were used for DNA sequencing.    Control libraries to test for contamination of CPDs followed the protocol described above, with the following modifications: WT cells were cultured and carried through to CPD photolyase treatment. Following this, samples were split in half, with half the samples treated with UVDE to cleave 6-4PP and TA-PP and the other half treated with T4 endo V to cleave any remaining CPD lesions. Following enzyme treatment, DNa was purified using phenol:chloroform:isoamyl alcohol and ethanol precipitation. Library preparation resumed with second (A) adapter ligation.