Schizosaccharomyces pombe
Protocols: S. pombe cells were inoculated in liquid YES medium overnight at 32°C, diluted to 2-5 x 10exp6 cells/ml the next morning and grown for 6 hours. Next, cells were resuspended in liquid EMM at 1 x 10exp8 cells/ml and 1 ml was treated or not with 20 microliters of EMS (ie 2% final) for 3h in 10 ml tubes at 32°C. Next, cells were washed three times with 5 ml of EMM, resuspended in 1 ml of EMM, plated on YES plates and incubated at 32°C until colonies formed. S. pombe spores were grown to saturation in 1.6 ml of YES medium at 32°C in 96 well plates (2 ml deep well plates). Cells were harvested by centrifugation, washed once with TE and lyzed during two hours at 37°C using 30 units of zymolyase in Y1 Qiagen buffer. Samples were centrifuged at 2,700g for five minutes and supernatants treated with 0.5 mg/ml of RNaseA for 10 minutes at room temperature. DNA was next purified from these lysates using the E-Z 96™ Tissue DNA Kit from Omega Bio-tek following the manufacturer's instructions except for DNA binding to the columns that was done by gravity flow instead of centrifugation. TPase-based DNA library (>95 samples)