mRNA stability study of Toxoplasma gondii in standard and low iron conditions

Human foreskin fibroblasts cells were pre-treated for 24 hours with 100uM deferroxamine (an iron chelator)before being infected with RH parasites. At 18 h post infection, 10 mg/ml Actinomycin D was added to the culture media at in the treatment dishes. Parasites were collected at 0, 1, 3 and 5 hours post-Actinomycin D treatment. At each collection, host cells were mechanically lysed and filtered through a 3mm membrane to remove debris. Parasites were pelleted by centrifugation and immediately frozen on dry ice for storage at -80°C. RNA was extracted using a Qiagen RNAeasy kit according to manufacturer's instructions. RNA libraries were then prepared using Illumina Stranded mRNA library preparation method and sequenced at 2 x 100 bp to an average of more than 10 million reads per sample.