|
| Status |
Public on Mar 19, 2009 |
| Title |
FR235222 treated RH strain, ChIP-on-chip acetylated H4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Toxoplasma gondii RH strain
|
| Organism |
Toxoplasma gondii |
| Characteristics |
agent: FR235222
fraction: IP
chip antibody: anti-acetyl histone H4 [acH4(K5-K8-12-K16)]
|
| Biomaterial provider |
HAKIMI MA and BOUGDOUR A
|
| Treatment protocol |
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.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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.chiponchip.org/index.html). NimbleGen's "Protocols for Chromatin Immunoprecipitation and Amplification" (http://www.vmrf.org/researchcenters/gene-chip/chromatin_immunoprecipitation.pdf).
|
| Label |
CY5
|
| Label protocol |
NimbleGen’s protocol (http://www.chiponchip.org/index.html). NimbleGen's "Protocols for Chromatin Immunoprecipitation and Amplification" (http://www.vmrf.org/researchcenters/gene-chip/chromatin_immunoprecipitation.pdf).
|
| |
|
| Channel 2 |
| Source name |
Toxoplasma gondii RH strain
|
| Organism |
Toxoplasma gondii |
| Characteristics |
agent: FR235222
fraction: input genomic DNA
|
| Biomaterial provider |
HAKIMI MA and BOUGDOUR A
|
| Treatment protocol |
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.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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.chiponchip.org/index.html). NimbleGen's "Protocols for Chromatin Immunoprecipitation and Amplification" (http://www.vmrf.org/researchcenters/gene-chip/chromatin_immunoprecipitation.pdf).
|
| Label |
CY3
|
| Label protocol |
NimbleGen’s protocol (http://www.chiponchip.org/index.html). NimbleGen's "Protocols for Chromatin Immunoprecipitation and Amplification" (http://www.vmrf.org/researchcenters/gene-chip/chromatin_immunoprecipitation.pdf).
|
| |
|
| |
| Hybridization protocol |
NimbleGen’s protocol (http://www.chiponchip.org/index.html).
|
| Scan protocol |
NimbleGen’s protocol (http://www.chiponchip.org/index.html).
|
| Description |
Toxoplasma gondii FR235222-treated parasite.
Immunoprecipitated DNA with the acH4(K5-K8-12-K16) antibody from FR235222 treated RH strain.
Input genomic DNA (from FR235222-treated RH strain)
|
| Data processing |
After background correction, log base 2 ratios of immunoprecipitated DNA signal over input DNA signal is given in this table. Data not normalized.
|
| |
|
| Submission date |
Mar 16, 2009 |
| Last update date |
Mar 18, 2009 |
| Contact name |
Mohamed-ali HAKIMI |
| E-mail(s) |
mohamed-ali.hakimi@univ-grenoble-alpes.fr
|
| Phone |
(33)476637469
|
| Organization name |
CNRS
|
| Department |
UMR5309
|
| Lab |
IAB - HAKIMI Team
|
| Street address |
Domaine de la Merci, Campus Santé
|
| City |
LA TRONCHE |
| State/province |
Grenoble |
| ZIP/Postal code |
38700 |
| Country |
France |
| |
|
| Platform ID |
GPL8305 |
| Series (1) |
| GSE15241 |
Drug-inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites |
|
