GSE119211
SFP1 (ChIP-seq)
We set out to determine how Sfp1 binding targets evolved over time. We sampled our species of interest to include one pre-whole genome duplicaton (WGD) species containing SFP1A (K. lactis), one post-WGD species containing both SFP1 and SFP1PL (N. castellii), two post-WGD species containing only SFP1 (S. cerevisiae and S. paradoxus) and performed ChIP for each Sfp1 homolog in each species using an antibody that recognizes a conserved subdomain of Sfp1 common to both Sfp1, Sfp1a, and Sfp1pl. Because Sfp1pl was also present in N. castellii, a second antibody was used that is more specific to Sfp1pl (denoted as IP3). We also performed ChIP in SFP1 deletion mutants in each species tested, as a control." Overall design: Polyclonal antibodies were created (Bethyl, Inc) to bind a peptide from the conserved c-terminal portion of Sfp1 designated, aSfp1-1, while aSfp1-3 was raised to recognize was raised to bind specifically to the N. castellii Sfp1pl The ChIP protocol was performed as described by Lefrancois et al. in The Guide to Yeast Genetics (Weissman, Jonathan F., Guthrie, 2010); modifications noted below) with 2-3 replicates per sample. Strains were grown to optical densities (OD600) between 0.6 and 0.8 in 500 mL of BMW (Thompson et al., 2013). Fixation took place with 37% formaldehyde for 15 minutes at room temperature, with occasional shaking every 5 minutes. Samples were sheared on the Covaris E210 with the following settings: 20% / 8 intensity/ 200 cycles per burst in 1-minute cycles for 16 minutes. Sequencing libraries were prepared from the input and immunoprecipitated DNA and were sequenced on an Illumina HiSeq 2500 with 25 base pair, paired end reads. Reads were mapped to the reference genomes of each species (S288C R64 for S. cerevisiae, YGOB v7 Aug 2012 for K. lactis and N. castellii, and Scannell et al. for S. paradoxus (Scannell et al., 2011), using bowtie2.