GSM8723841: SDL-TOPO library prepared from 25 ng of genomic DNA extracted from cdc9-7 mutant of budding yeast cells; Saccharomyces cerevisiae; OTHER
Cells were collected with centrifugation, washed once with water, and served for genomic DNA extraction using Genomic-tip 100/G from Qiagen following the yeast-specific protocol provided by the manufacturer. A 14.5 μL reaction containing the indicated amount of genomic DNA, 2.5 μL 10x SDL buffer, 1 μL rSAP (NEB) was prepared and incubated at 37°C for 15 min and 95°C for 5 min. Then, the reaction was supplemented with 7.5 μL PEG600, 1 μL each 10 mM ATP, 10 μM P-AR-thio5-NH2, and 5 mg/ml SDL. The reaction was incubated at 37°C for 1 h and 95 °C for 5min. Subsequently, the solution was supplemented with 15 µL of water, 5 µL of 10×PCR buffer (Takara Bio Inc.), 4 µL of 2.5 mM dNTPs, 1 µL of 50 µM NH2-AF (Supplementary Table S1), and five units of GeneTaq hot start (Nippon gene, Toyama, Japan) and incubated sequentially at 95°C for 3 min, 55°C for 5 min, and 72°C for 5 min. Then, 50 µL of AxyPrep Mag PCR clean and 50 µL of isopropanol were added and incubated at 5 min. The beads were washed twice with 200 µL of the solution containing 19% (v/v) PEG #400, 1 M NaCl, and 50 mM Tris-HCl, pH 8.0, and rinsed once with 70%(v/v) ethanol. The purified DNA was eluted with 25 µL of 10 mM Tris-Acetate, pH 8.0. To the purified DNA solution, 15 µL of water, 5 µL of 10×ExTaq buffer, 4 µL of 2.5 mM dNTPs, 1 µL of ExTaq HS, 1 µL of 20 µM Primer-3, 1 µL of 20 µM Index primer, and 1 µL of 5 ng/µl VOC for TA were added. After sequential incubation at 25°C for 5 min, 72°C for 5 min, and 95°C for 3 min, appropriate cycles of three-step incubations for 15 s at 95°C, 55°C, and 72°C, and a final extension at 72°C for 1 min were performed.