GSM4212296: Ste12_K152L_variant rep 2; Saccharomyces cerevisiae; OTHER
Cells were collected from selection plates and genomic DNA was extracted from each sample using the Smash and Grab method as described (Hoffman and Winston 1987). Each DNA sample was divided into two 20-μg aliquots and digested by TaqI and RsaI (NEB) individually. Digested products were purified by DNA purification column (Zymo Research) and ligated overnight at 15°C in dilute solution to encourage self-ligation. Self-ligated DNA was purified by DNA purification column (Zymo Research) and used as template in an inverse PCR. Primers that anneal to the PiggyBac donor sequences (primer 316: GATGTCCTAAATGCACAGCGAC and primer 317: GAGGCGTGCTTGTCAATGC) were used to amplify the genomic regions flanking the transposon, and then adaptor sequences that allow the PCR products to be sequenced on Illumina sequencing platforms were added by primer 367 (AATGATACGGCGACCACCGAGATCTACACCTCCATCGAGACACTCTTTCCCTACACGACG CTCTTCCGATCTCGTCAATTTTACGCAGACTATC) and 377 (CAAGCAGAAGACGGCATACGAGATTGCTTGTCAATGCGGTAAG). The PCR products were purified using a DNA purification column (Zymo Research). For each sample, the same amount of PCR product from digestion with each restriction endonuclease was pooled and submitted for Illumina sequencing.
GEO
GSM4212296
GEO Accession
GSM4212296