GSM7412382: Point mutant Matα2, High selection; Saccharomyces cerevisiae; OTHER
In each case, cells were pelleted by centrifugation, washed in PBS, and the pellets frozen at -80°C. Subsequent extraction of plasmid DNA was accomplished by thawing and boiling the cells for 5 minutes followed by bead-beating in the presence of phenol and chloroform. DNA was ethanol precipitated and purified using a QIAGEN QIAprep miniprep kit. Sequencing libraries were prepared using PCR to amplify the variable region of MATα2 from the plasmid pool using primers containing Illumina adapter sequences and sample specific barcodes. For each sample/library, ten 50 µL PCR reactions were pooled after nine PCR cycles. The 500 µL PCR pool was cleaned and concentrated using a QIAGEN MinElute PCR Purification Kit and quantified using either an Agilent Bioanalyzer or TapeStation (High sensititivity DNA kit).