title: GSM3061026: WT.oxoG.rep2; Saccharomyces cerevisiae BY4741; OTHER
library_construction_protocol: Genomic DNA from S. cerevisiae cells was grown overnight in liquid rich medium containing yeast extract, peptone and dextrose (YPD) , then extracted with a DNA Isolation Kit for Cells and Tissues (Roche Holding AG) according to the manufacturer's instructions. To minimize adventitious 8-oxoG formation, antioxidants (100 mM deferoxamine, 100 mM butylated hydroxytoluene) were used until the 8-oxoG excision step. Genomic  DNA (5 μg) was sheared in 130 μl Tris-EDTA buffer with a Covaris S220 ultrasonicator (Covaris Inc.) The DNA fragments were treated with APE 1 (final concentration (fc): 0.2U/μl) in 1 × NEBuffer 2.1 at 37 °C for 1 h to remove abasic sites. Dideoxynucleotides (Jena Bioscience GmbH, fc: 200 μM) and therminator IX (NEB, fc: 0.03 U/μl) were added and heated (60 °C, 10 min) to block gaps and terminal nucleotides in DNA fragments. The product was purified using the Monarch nucleic acid purification kit (NEB). The removal of 8-oxoG was carried out with 1 μl Fpg (NEB, 8 U/ μl) and 1 μl APE 1 (NEB, 10 U/μl) in 50 μl 1 × NEBuffer 2.1 at 37 °C for 1 h. The reaction mixture was then heated (60 °C, 10 min) to inactive Fpg and APE1. After allowing to return to room temperature, 1 μl of prop-dGTP (10 mM) and 0.2 μl of therminator IX (NEB, 10 U/ μl) was added and the mixture was heated (60°C, 10 min).The resulting DNA was purified with nucleic acid purification kit, completely dried on a vacuum concentrator and re-suspended in 4 μl water. Next, 6 μl L19-azido (200 μM), 2 μl potassium phosphate buffer (1 M, pH 7.0), 2 μl aminoguanidine hydrochloride solution (50 mM), 2 μl DMSO, 2 μl sodium ascorbate (25 mM) and 2 μl premixed CuSO4:THPTA (1:6, 5 mM in concentration of Cu2+) were add and incubated at room temperature (30 min). After purification, DNA (50 μl in TE buffer) was heated ( 95 °C,  2 min) and the tube was quickly transfered to an ice bath. DNA was subjected to slow rotation with Dynabeads MyOne C1 (Invitrogen Corp.) in bead-binding buffer (5 μl, 5.0 mM Tris-HCl (pH 7.5), 1.0 mM EDTA, 1 M NaCl) at room temperature. for 30 min. Afterwards, the beads were washed twice with bead-washing buffer (200 μl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.2 M NaCl) and resuspended in 49 μl reaction mixture (5 μl T4 PNK reaction buffer (10 ×), 5 μl ATP (10 mM) and 39 μl ddH2O). After adding 1 μl T4 PNK (NEB), the mixture was warmed at 37 °C for 30 min. Next, the beads were washed two times with 200 μl bead-washing buffer and resuspended in 49 μl reaction mixture (5 μl T4 ligase reaction buffer (10 ×), 5 μl PEG-4000 (50 %), 0.5 μl TWEEN-20 (1 %), 3 μl pre-annealed dsDNA adapter (40 μM) ). After adding 1 μl T4 ligase (NEB), the mixture was incubated at 16 °C for 2 h. After adaptor ligation, the beads were washed twice with 200 μl bead-washing buffer, resuspended in 20 μl ddH2O and heated at 95 °C for 2 min. The beads were pelleted immediately using a magnetic rack, and the supernatant, which contained the single-stranded library molecules, was transferred to a fresh tube. The entire library was used for indexing with one of the barcoded primers, P7-index1-3, and Ex-P5 using Vent exo- polymerase (NEB) in 1 × Thermopol buffer. The indexed library was purified using an SPRIselect kit (Beckman Coulter) according to the manufacturer's instructions. After indexing, the library was amplified with Pr-P5-2nd and Pr-P7-2nd using Q5 high fidelity DNA polymerase (NEB) and purified with an SPRIselect kit. The library size was verified using the Agilent 2200 tapestation with high sensitivity D1000 screen tape before being loaded onto the Illumina Miseq platform (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM3061026