|Number of samples:||2|
|Release date:||Mar 19 2009|
|Last update date:||Mar 21 2012|
|Dataset link||Drug-inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites|
Freshly released tachyzoites were needle-passed, and filtered using a 3-µm nucleopore membrane. Parasites were resuspended into fresh DMEM supplemented with 10% (v/v) FBS and 25 mM HEPES buffer pH7.2. Parasites were incubated in the presence of FR235222 (40 nM) or DMSO (0.1%) for 4 h at 37°C with 5% CO2. For ChIP-chip experiments freshly released tachyzoites (~5 x 109 at ~12 x 107 parasites/mL) were fixed for 15 min in 1% formaldehyde. Increase in AcH4 signals was verified by immunoblot to verify that FR235222 treatment was effective. To prepare chromatin samples, fixed parasites were lysed in MNase buffer (0.32 M Sucrose, 50 mM Tris-HCl pH7.8, 4 mM MgCl2, 3 mM CaCl2, 100 mM NaCl, 0.25% (v/v) NP40, 5% (v/v) glycerol, protease inhibitor EDTA-free cocktail (Roche)) and DNA was digested for 4 min at 37°C by MNase (2 units/mL). Digestion was stopped with 20 mM EDTA and chromatin was recovered in the soluble fraction after centrifugation at 10,000 g at 4°C; this constituted the S1 fractions. Pelleted materials were resuspended in dialysis buffer (1mM Tris-HCl pH7.8, 0.2 mM EDTA) containing 1 mM PMSF and protease inhibitor cocktail (Roche®) and dialyzed overnight at 4°C against the same solution. Then dialyzed materials were centrifuged and supernatant were harvested; this constitutes the S2 fractions. For chromatin immunoprecipitations, fractions S1 and S2 were pooled and DNA quality was verified by electrophoresis on 2% agarose gels; oligonucleosome ladder of 100-1000 bp were obtained. The histone-DNA complexes were immunoprecipitated with anti-acetyl histone H4 (Upstate®, catalog # 06-866) antibodies according to NimbleGen’s protocol (http://www.genomecenter.ucdavis.edu/expression_analysis/documents).
Citations per year
The Journal of Experimental Medicine