title: GSM4490228: wt_NoUV A1; 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/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 5 μg of DNA was used for each sequencing library preparation. CPD-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 T4 endonuclease V and apurinic/apyrimidinic (AP) endonuclease, APE 1, to generage new 3'-OHs. DNA was then ligated to adaptor  A. Up to 3 different barcoded A adaptors (A1: AAGAGGAT; A2: TTCGTGAT; A3: CCTGAGAT) were used to generate libraries for different experiments. DNA was purified with Streptavidin beads and amplified for up to 6 cycles in a PCR reaction with primers complimentary to A and trP1, and the resulting DNA libraries were used for DNA sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4490228