GSM5731052: C_R3; Schizosaccharomyces pombe; OTHER
For plasmid library from E.coli, use QIAfilter Plasmid Giga Kit (Qiagen) following manufacturer's manual. For DNA extracted from S.pombe, cells (~1.5x109)  were harvested and washed once with ddH2O and once with Z buffer (50 mM sodium citrate, 50 mM sodium phosphate dibasic, 40 mM EDTA at pH 7.8) followed by incubation in 2 mL of Z buffer plus 0.5 mg/mL Zymolase T100 (US Biological) and 2 mM dithiothreitol for 1 h at 37°C. Sodium dodecyl sulfate was then added to a final concentration of 2% (w/v), and incubation was continued for 10 min at 65°C. The volume was then increased to 10 mL with 5× TE (50 mM Tris-HCl at pH 8.0, 5 mM EDTA), and proteinase K (Sigma-Aldrich) was added to 50 μg/mL. After 1 h of incubation at 50°C, samples were precipitated with 3 mL of 5 M potassium acetate for 30 min on ice, and the precipitate was removed by two rounds of centrifugation at 3200g for 10 min. The supernatant was then mixed with 1 vol of 100% isopropanol. After 20 min on ice, DNA was collected by centrifugation at 10,500g for 10 min and resuspended in 5× TE with 50 μg/mL RNase A. After 1 h of incubation at 37°C, DNA was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1, equilibrated with 5× TE) and once with chloroform:isoamyl alcohol (24:1, equilibrated with 5× TE) and then precipitated for 1 h at −20°C following the addition of 2.5 vol of ethanol. DNA was collected by centrifugation at 16,000g for 10 min followed by one wash with 70% ethanol. DNA was solubilized in 1× TE buffer. To amplify the mutated region for Illumina sequencing, PCR reactions (50 µl) containing 1 x Q5 reaction buffer (NEB), 200 µM dNTPs, 0.5 µM of Bloli6118 (TTCTTGATTGTAAAGTAAATTAC) and Bloli6119 (TAGAGAAATAGAATTTGGGC) , 1 U of Q5 Hot Start High-Fidelity DNA Polymerase (NEB) and 100ng genomic DNA from R0-R5 or 93pg of the original plasmid library isolated from bacteria were incubated at 98°C for 1min, followed by 18 cycles of 98°C for 10s, 56°C for 30s and 72°C for 30s with a final extension at 72°C for 2min. PCR products were gel purified with MinElute gel purification kit (Qiagen) and Illumina libraries were prepared with different indexes using Nextera XT Library Prep Kit (Illumina, FC-131-1096).
GEO Accession
GSM5731052