title: Sequencing data of PCR on downtag barcode at ARS404 of Pol2 ChIP samples (Repli-ID) in Saccharomyces cerevisiae
design: The strain containing myc-tagged Pol-epsilon  (Pol2-9xMyc) and the galactose-inducible sld3-38A dbf4-4A construct (adapted from (Zegerman & Diffley, 2010)) integrated at BAR1 locus were crossed into barcoded libraries to generate the Repli-ID library. Library plates were grown overnight on YPAD plates containing raffinose at 30C. In the morning, they were diluted to 0.1 OD and grown for 3 hours in YPAD containing raffinose at 30C. To arrest the cells in G1, alpha-factor was added for 2.5 hours with extra additions after 1 hour and after 2 hours. During the last 30 minutes of alpha-factor arrest, galactose was added to induce expression of the SLD3/DBF4 mutant construct and subsequently fire all origins during release into S-phase. Alpha-factor was removed by washing the yeast twice in YPAD and released into YPAD containing 0.2M HU (Sigma-Aldrich) and 50 g/ml pronase (Merck Millipore). Samples were crosslinked using 1% formaldehyde after 40 and 80 minutes. ChIP was performed as in Vlaming et al. (2016), except that it was performed using the anti-myc antibody. DNA was isolated and PCR amplified using Downtag primers as described in Vlaming et al. (2016). PCR products were deep-sequenced on a single-end flow-cell Illumina Hi-Seq2500. Barcodes were counted and depletion or enrichment of a barcode was scored as in Vlaming et al. (2016).