GSM2585804: newmag1 0hr; Saccharomyces cerevisiae; OTHER
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/T1 (ThermoFisher Scientific) at 37˚C for 1 hr to remove RNA. DNA was subsequently purified with PCI extraction, ethanol precipitated, and disolved in sterile deionized H2O. The purified genomic DNA was quantified and 12 μg of DNA was used for each sequencing library preparation. NMP-seq procedure is adopted from the recently published emRiboSeq method (Nature Protocols 2015 10, 1433-1444). 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. CPD lesions were cleaved with Alkyladenine DNA glycosylase (AAG) and apurinic/apyrimidinic (AP) endonuclease, APE 1, to generage new 3'-OHs. 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 time points. DNA was purified with Streptavidin beads and amplified for 5-6 cycles in a PCR reaction with primers complimentary to A and trP1, and the resulting DNA libraries were used for DNA sequencing.
GEO Accession
GSM2585804