TrAEL-seq for CUP1 locus

1) The Pgal-3HA cup1 yeast strain, in which all CUP1 ORFs are replaced with 3HA and all CUP1 promoters are replaced by GAL1-10 promoters (PMID: 28654659, which carries ~17 tandem repeats of the modified Pgal-3HA cup1 repeat) undergoes extensive CNV when shifted to galactose. Here we use TrAEL-seq to look for replication fork stalling in this strain with and without induction. 2) We also profile the impact on replication of integrating an ARSH4 origin upstream of the RSC30 gene (so outside the CUP1 locus), using the 3xCUP1 strain which carries 3 repeats of CUP1 (PMID: 28654659). The origin was integrated with a URA3 marker, the control strain has the URA3 marker but no origin. 1) Cells were pre-grown on YPraffinose, then resuspended in fresh YP 2% raffinose supplemented with nothing, 0.02% galactose, 2% galactose or 2% glucose as specified. Cells were harvested after 5 hours by resuspension in 70% ethanol. 2) Cells were grown in SC glucose and harvested by re-suspension in 70% ethanol.