Chromatin purification, immunoprecipitation, quantitative real-time PCR (qPCR) amplification and data analysis were performed as described (44 (link),50 (link),51 (link)). Briefly, cells were grown overnight to saturation at 25°C, then diluted to an OD600 of ∼0.2, and let them grow at 34 °C for ∼5 h until they reached an OD600 of 0.5–0.6. Immunoprecipitated and purified chromatin was subjected to quantitative real-time PCR using the CFX96 Detection System (Bio-Rad Laboratories, Inc.) and SYBR® Premix Ex Taq™ (Takara Bio), following the manufacturer's instructions. Real-time PCR reactions were performed in duplicate from at least three independent ChIPs. Quantitative analysis was performed with the CFX96 Manager Software (version 3.1, Bio-Rad). The values obtained for the IPed PCR products were compared to those of the total input, and the ratio of the values from each PCR product from transcribed genes to a non-transcribed region of chromosome VII was calculated. Numbers on the y-axis of graphs are detailed in the corresponding figure legend.
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Quantitative Chromatin Immunoprecipitation and qPCR
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Corresponding Organization : Clermont Université
Other organizations : Universitat de València
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Variable analysis
independent variables
- Cell growth temperature (25°C overnight, then 34°C for ~5 h)
- Chromatin immunoprecipitation (ChIP)
- Quantitative real-time PCR (qPCR) amplification
- Ratio of IPed PCR products from transcribed genes to a non-transcribed region
- Cell density (OD600 of ~0.2, then 0.5-0.6)
- Quantitative real-time PCR performed in duplicate from at least three independent ChIP samples
- Not explicitly mentioned
- Comparison of IPed PCR products from transcribed genes to a non-transcribed region of chromosome VII
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