Chip-Seq with HapX in Paracoccidioides brasiliensis Pb18 - rep3
P. brasiliensis yeast cells were grown in MVM liquid medium containing 50 M BPS for 24 hours to induce iron deficiency and the expression of HapX. For cross-linking, 1% v/v formaldehyde was used, followed by incubation for 20 minutes at 37C with shaking. Cross-linking was stopped by the addition of 125 mM glycine, followed by incubation for 10 minutes under the same conditions as before. For chromatin immunoprecipitation (ChIP), cells were washed, frozen in liquid nitrogen and lyophilized for 3 hours. The lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS) and beads were used for mechanical fragmentation with a bead beater followed by sonication. Co-immunoprecipitation was performed with the Dynabeads Immunoprecipitation Kit according to the manufacturer's instructions. The anti-HapX antibody was used to bind the target fragmented DNA. Briefly, the anti-HapX antibody was bound to the beads and then incubated with the fragmented DNA sample, after which the antibody and the interacting DNA were eluted from the beads. Three replicates of ChIP-seq libraries were constructed and sequencing was performed in the Illumina NovaSeq6000 platform.