A student interested in the regulation of facultative heterochromatin needs to optimize the experimental conditions of her chromatin immunoprecipitation (ChIP) assay using heart tissue. For this, she crosslinks mouse heart tissue with 1% formaldehyde for either 10 or 30 minutes. The student then performs ChIP with the RING1B and SUZ12 antibodies. RING1B and SUZ12 belong to the Polycomb Repressor complex 1 (PRC1), which maintains genes silenced in specific tissues. The enriched DNA is quantified by real-time PCR using primers to the indicated genes. The amount of immunoprecipitated DNA in each sample is represented as normalized signal to the negative locus GAPDH, which is equal to 1.A. Why does the student consider GAPDH a negative locus, i. e. where RING1B and SUZ12 do not bind?
B. What is the transcriptional status of the HoxD10 and HoxA1 genes in the heart? In one sentence explain your reasoning.
C. If you were this student, which fixation condition (10 or 30 min) would you use? In one sentence explain your reasoning.