title: ChIP-seq of Cg wild-type untagged3
design: C. glabrata wild-type (untagged) and Pdc2-myc tagged strains, as well as a CgThi3-myc tagged strain, were grown at 30o C in SD complete medium to logarithmic growth phase.  The cells were harvested, washed with sterile water, inoculated (in triplicate) in SD medium with and without thiamine, and grown overnight at room temperature (20o C) to an OD of ~1.20. The DNA binding proteins were cross-linked with 1% formaldehyde for 15 minutes and quenched with 2.5 M glycine for 10 minutes, both at room temperature (20o C). The cells were then washed with PBS, frozen with liquid nitrogen, and stored at -80o C. Cells were lysed by bead beating in lysis buffer (50 mM HEPES-KOH, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) and chromatin was sheared by sonication. Lysate was incubated with 2 g c-Myc monoclonal antibody (9E10.3; Invitrogen) at 4 C for 2 hours while gently rotating. Antibody bound protein-DNA complexes were immunoprecipitated using Dynabeads Protein G (Invitrogen) and eluted using ChIP elution buffer (50 mM Tris/HCl, 10 mM EDTA, 1% SDS). Crosslinks were reversed and the DNA was purified using a spin column (Zymo Research DNA Clean and Concentrator Kit). Enrichment of C. glabrata Pdc2 and Thi3 at promoters during thiamine starvation was quantified using next generation sequencing. Illumina Miseq libraries were generated using the immunoprecipitated DNA from both myc-tagged Pdc2 and Thi3 strains and the untagged wild-type strain (NEBNext Ultra II for DNA Library Prep kit for Illumina, New England Biolabs).