GSM1846283: Induced_presort control_dntag_replicate_2; Saccharomyces cerevisiae; OTHER
The sorted cells were grown to saturation in 15ml SC–Leu, spun down, and stored as glycerol stocks at –80°C. ~25 ODs of cells were used for gDNA extraction. The cells were first thawed at room temperature and resuspended in 250µl of reconstitution buffer (1ml of resuspension solution concentrate, 0.18g sorbitol and 1µl beta-mercaptoethanol). 1000 Units of reconstituted lyticase enzyme were added to each tube, and each tube was mixed by inversion a few times. The tubes were incubated at room temperature for 1 hour with gentle mixing by inversion every 15 minutes. To isolate the gDNA, the Norgen Biotek Yeast Genomic DNA Isolation Kit (Product #18600) was used. The UPTAG and DNTAGS were amplified with primers (sequences available in supplemental methods). Each PCR reaction was performed in a final volume of 100ul, containing 1 Unit of Phusion polymerase, HF phusion buffer diluted to 1X, 250 µM dNTP, 250 nM of each primer and ~100ng genomic DNA. The PCR amplification protocol used was: 30 sec at 98°C; 25 cycles of 10 sec at 98°C, 30 sec at 55°C, 30 sec at 72°C; 7 min at 72°C; ∞ at 4°C.. The PCR reactions were run on a 2% agarose gel, gel extracted using the ZymocleanTM Gel DNA Recovery Kit, pooled in equimolar amounts and sent for single-end, 50-cycle sequencing on an Illumina HiSeq 2500.