Figure 3

Generation of the barcode-tagged S. pombe insertion mutant library. The linear insertion DNA (Figure 1B) was transformed into the wild type strain KRP1 to obtain Ura⁺ transformants on minimal medium (MMA) with multiple nutritional supplements except uracil (YC – uracil) and low levels of 5-FOA (0.1 g/l). Transformants were then tested for stable integration by 5-FOA sensitivity. Stable transformants (i.e. 5-FOA sensitive cells) were inoculated in non-selective YES medium in 96-well plates, followed by assembling four such plates on a synthetic medium plate lacking uracil (EMM + YC – uracil) and a similar medium plate that contains uracil and 1 g/l of 5-FOA (EMM + YC + 5-FOA) to generate 384-colony arrays for the second 5-FOA sensitivity test. Unstable transformants found in this second screen were removed before these mutants were stored as 384-well mutant arrays or mixed mutant pools of ~1800 mutants.