GSM3207839: ChIP.rep3; Cryptococcus neoformans var. grubii H99; ChIP-Seq
ChIP assay: The ChIP assay was performed using the ChIP-IT High Sensitivity Kit (Active Motif, Carlsbad). The DAC4-FLAG strain cells were diluted in fresh YPD medium. Cells were fixed with 1% formaldehyde (Sigma, USA) for 15 mins and quenched by 125 mM glycine. Cells were washed three times with ice-cold PBS buffer supplemented with 125 mM glycine. Cells were then lysed with the lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.5% IGEPAL). Cell pellets were suspended in sonication buffer (20 mM Tris-HCL (PH 8.0), 2 mM EDTA, 1% Triton-X100, 150 mM NaCl, 1% SDS, 100 µM PMSF and 1x proteinase inhibitor). The samples were sheared with a Diagenode Pico device using 30 s on and off for 30 cycles. After centrifugation, the supernatant was collected and diluted in IP dilution buffer (20 mM Tris-HCl, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, 100 μM PMSF and 1x proteinase inhibitor). The immunoprecipitation was performed with 5 μg of anti-Flag ChIP grade antibody and Protein G Beads at 4°C overnight. The beads were then washed 4 times in IP dilution buffer. The sample was then eluted with elution buffer and treated with Proteinase K overnight at 65°C. DNA samples were purified with DNA purification columns. The ChIP-seq library was constructed with the MicroPlex Library Preparation Kit v2 (Diagenode, Liège, Belgium) according to the manufacturer's instructions.